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The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.

Class A scavenger receptors (SR-A) are transmembrane glycoproteins that mediate both ligand internalization and cell adhesion. Previous studies have identified specific amino acids in the cytoplasmic tail of SR-A that regulate receptor internalization; however, the role of cytoplasmic domains in regulating cell adhesion has not been addressed. To investigate the role of cytoplasmic domains in SR-A-mediated adhesion and to address whether SR-A-mediated adhesion and internalization require distinct cytoplasmic domains, different SR-A constructs were stably expressed in human embryonic kidney (HEK 293) cells. Deleting the entire cytoplasmic tail (SR-A Delta 1-55) greatly reduced receptor protein abundance. Retaining the six amino acids proximal to the membrane (SR-A Delta 1-49) restored receptor protein abundance. Although SR-A Delta 1-49 localized to the cell surface, cells expressing this receptor failed to internalize the ligand acetylated low density lipoprotein. Replacing the cytoplasmic tail of SR-A with that of the transferrin receptor (TfR/SR-A) resulted in retention of the chimeric receptor in the endoplasmic reticulum suggesting a specific role for the membrane-proximal amino acids in trafficking SR-A from the endoplasmic reticulum to the Golgi. Like SR-A expressing cells, cells expressing SR-A Delta 1-49 displayed increased spreading and adhesion, demonstrating that the membrane-proximal amino acids were sufficient for SR-A-mediated cell adhesion. Together, our results indicate a critical role for the membrane-proximal amino acids in SR-A trafficking and demonstrate that SR-A-mediated adhesion and internalization require distinct cytoplasmic domains.

Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13-(alpha) and 40 (beta)-kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using 14C- and BODIPY-labeled C12-ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly "high mannose" type oligosaccharides and that two distinct beta-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the approximately 2-4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using 14C-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be co-precipitated with acid ceramidase using anti-ceramidase antibodies. Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase.

Bone morphogenetic protein (BMP)-1 is a zinc-dependent metalloproteinase that cleaves a variety of extracellular matrix substrates, including type I procollagen. Little is known about the site of action of BMP-1, although the extracellular matrix seems likely to be it. BMP-1 is synthesized with an N-terminal prodomain. The removal of the prodomain presumably activates the proteinase. In this study we show that the prodomain is cleaved in the trans-Golgi network (TGN) and by furin-like/paired basic proprotein convertases. Inhibitors of furin resulted in the secretion of pro-BMP-1, which could not cleave procollagen. Recombinant furin cleaved the prodomain from pro-BMP-1. Site-directed mutagenesis of the prodomain cleavage site (RSRR) to RSAA resulted in efficient secretion of pro-BMP-1. Therefore, prodomain cleavage was not required for secretion. Using peptide N-glycosidase and neuraminidase digestion to determine the post-translational status of pro-BMP-1 during its conversion to BMP-1, we showed that BMP-1 first appears in the TGN during sialylation of the molecule. Furthermore, immunofluorescence studies using an antibody to the nascent N terminus of BMP-1 showed localization to the TGN and plasma membrane. The observation that BMP-1 occurs inside the cell raises the possibility that BMP-1 might begin to cleave its substrates prior to secretion to the extracellular matrix.

The neural cell adhesion molecule and the voltage-sensitive sodium channel alpha-subunit are the only two molecules in mammals known to be modified by alpha-2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide:N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.

YY1 is a zinc finger DNA-binding transcription factor that influences expression of a wide variety of cellular and viral genes. YY1 is essential for the development of mammalian embryos. It regulates the expression of genes with important functions in DNA replication, protein synthesis, and cellular response to external stimuli during cell growth and differentiation. How YY1 accomplishes such a variety of functions is unknown. Here, we show that a subset of the nuclear YY1 appears to be O-GlcNAcylated regardless of the differentiation status of the cells. We found that glucose strongly stimulates O-linked N-acetylglucosaminylation (O-GlcNAcylation) on YY1. Glycosylated YY1 no longer binds the retinoblastoma protein (Rb). Upon dissociation from Rb, the glycosylated YY1 is free to bind DNA. The ability of the O-glycosylation on YY1 to disrupt the complex with Rb leads us to propose that O-glycosylation might have a profound effect on cell cycle transitions that regulate the YY1-Rb heterodimerization and promote the activity of YY1. Our observations provide strong evidence that YY1-regulated transcription is very likely connected to the pathway of glucose metabolism that culminates in the O-GlcNAcylation on YY1, changing its function in transcription.

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.

Despite numerous reports suggesting that beta(1) integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that beta(1) integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the beta(1) integrin subunit becomes hyposialylated, suggesting that the beta(1) integrin is a substrate for this enzyme. The expression of hyposialylated beta(1) integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified alpha(5)beta(1) integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated beta(1) integrins are more adhesive to fibronectin, although desialylation of alpha(5) subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the beta(1) integrin family of cell adhesion receptors.

MARCO is a type II transmembrane protein of the class A scavenger receptor family. It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain. Previous studies have indicated a role for this receptor in anti-microbial host defense functions. In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties. The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system. Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure. Rotary shadowing also demonstrated that the molecules often associate with each other via the globes. sMARCO was found to bind avidly both heat-killed and living bacteria. Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO. Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition. Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied. Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity. Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.

We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.

Testicular Sertoli cells phagocytose apoptotic spermatogenic cells in a manner depending on the membrane phospholipid phosphatidylserine (PS) expressed at the surface of the latter cell type. Our previous studies have indicated that class B scavenger receptor type I (SR-BI) is responsible for the PS-mediated phagocytosis by Sertoli cells. We examined here whether SR-BI binds directly to PS. A cell line acquired the ability to bind to PS-exposing apoptotic cells and to incorporate PS-containing liposomes when it was forced to express SR-BI. Furthermore, the extracellular domain of rat SR-BI fused with human Fc (SRBIecd-Fc) bound to PS with a dissociation equilibrium constant of 2.4 x 10(-7) m in a cell-free solid-phase assay, whereas other phospholipids including phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine were poor binding targets. The binding activity was enhanced when CaCl(2) was included in the assay or when SRBIecd-Fc was pre-treated with N-glycanase. A portion of the extracellular domain spanning amino acid positions 33 and 191 (numbered with respect to the amino terminus) fused with Fc (SRBI33-191-Fc) showed activity and phospholipid specificity equivalent to those of SRBIecd-Fc. Finally, SRBI33-191-Fc bound to the surface of apoptotic cells with externalized PS, and the injection of SRBI33-191-Fc into the seminiferous tubules of live mice increased the number of apoptotic spermatogenic cells. These results allowed us to conclude that SR-BI is a phagocytosis-inducing PS receptor of Sertoli cells.

Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases.

In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome. We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p. Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses. The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities.

In this study we have explored the endoplasmic reticulum associated events accompanying the maturation of the tyrosinase-related protein-1 (TRP-1) nascent chain synthesized in mouse melanoma cells. We show that TRP-1 folding process occurs much more rapidly than for tyrosinase, a highly homologous protein, being completed post-translationally by the formation of critical disulfide bonds. In cells pretreated with dithiothreitol (DTT), unfolded TRP-1 is retained in the endoplasmic reticulum by a prolonged interaction with calnexin and BiP before being targeted for degradation. The TRP-1 chain was able to fold into DTT-resistant conformations both in the presence or absence of alpha-glucosidase inhibitors, but folding occurred through different pathways. During the normal folding pathway, TRP-1 interacts with calnexin. In the presence of alpha-glucosidase inhibitors, the interaction with calnexin is prevented, with TRP-1 folding being assisted by BiP. In this case, the process has similar kinetics to that of untreated TRP-1 and yields a compact form insensitive to DTT as well. However, this form has different thermal denaturation properties than the native conformation. We conclude that disulfide bridge burring is crucial for the TRP-1 export. This suggests that although various folding pathways may complete this process, the native form may be acquired only through the normal unperturbed pathway.

Amyloid beta-peptide is generated by two sequential proteolytic cleavages mediated by beta-secretase (BACE) and gamma-secretase. BACE was recently identified as a membrane-associated aspartyl protease. We have now analyzed the maturation and pro-peptide cleavage of BACE. Pulse-chase experiments revealed that BACE is post-translationally modified during transport to the cell surface, which can be monitored by a significant increase in the molecular mass. The increase in molecular mass is caused by complex N-glycosylation. Treatment with tunicamycin and N-glycosidase F led to a BACE derivative with a molecular weight corresponding to an unmodified version. In contrast, the mature form of BACE was resistant to endoglycosidase H treatment. The cytoplasmic tail of BACE was required for efficient maturation and trafficking through the Golgi; a BACE variant lacking the cytoplasmic tail undergoes inefficient maturation. In contrast a soluble BACE variant that does not contain a membrane anchor matured more rapidly than full-length BACE. Pro-BACE was predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurred immediately before full maturation and trafficking through the Golgi.

Endoglycoceramidase (EC ) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here the purification, characterization, and cDNA cloning of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 and was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxycholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to hydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid sequence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Glu-Pro sequence, which is the putative active site of Rhodococcus endoglycoceramidase, was conserved in the deduced amino acid sequences. This is the first report of the cloning of an endoglycoceramidase from a eukaryote.

GIRK (G protein-gated inward rectifier K(+) channel) proteins play critical functional roles in heart and brain physiology. Using antibodies directed to either GIRK1 or GIRK4, site-directed mutagenesis, and specific glycosidases, we have investigated the effects of glycosylation in the biosynthesis and heteromerization of these proteins expressed in oocytes. Both GIRK1 and GIRK4 have one extracellular consensus N-glycosylation site. Using chimeras between GIRK1 and GIRK4 as well as a GIRK1 N-glycosylation mutant, we report that GIRK1 was glycosylated at Asn(119), whereas GIRK4 was not glycosylated at Asn(132). GIRK1 membrane-spanning domain 1 was required for optimal glycosylation at Asn(119) because a chimera that contained GIRK4 membrane-spanning domain 1 significantly reduced the addition of a carbohydrate structure at this site. This finding may partly account for the reason that GIRK4 is not glycosylated at Asn(132), either as a homomer or when coexpressed with GIRK1. When the GIRK1(N119Q) mutant was coexpressed with GIRK4, the biophysical properties of the heteromeric channel and the magnitude of the agonist-induced currents were similar to those of controls. Thus, N-glycosylation of GIRK1 at Asn(119) does not appear to affect its physical association with GIRK4, the routing of the heteromer to the cell surface, or heteromeric channel function, unlike the dramatic functional effects of N-glycosylation of ROMK1 at Asn(117) (Schwalbe, R. A., Wang, Z., Wible, B. A., and Brown, A. M. (1995) J. Biol. Chem. 270, 15336-15340).

In mammals, the Rh family includes the variable Rh polypeptides and invariant RhAG glycoprotein. These polytopic proteins are confined to the erythroid lineage and are assembled into a multisubunit complex essential for Rh antigen expression and plasma membrane integrity. Here, we report the characterization of RhCG and Rhcg, a pair of novel Rh homologues present in human and mouse nonerythroid tissues. Despite sharing a notable similarity to the erythroid forms, including the 12-transmembrane topological fold, the RHCG/Rhcg pair is distinct in chromosome location, genomic organization, promoter structure, and tissue-specific expression. RHCG and Rhcg map at 15q25 of human chromosome 15 and the long arm of mouse chromosome 7, respectively, each having 11 exons and a CpG-rich promoter. Northern blots detected kidney and testis as the major organs of RHCG or Rhcg expression. In situ hybridization revealed strong expression of Rhcg in the kidney collecting tubules and testis seminiferous tubules. Confocal imaging of transiently expressed green fluorescence protein fusion proteins localized RhCG exclusively to the plasma membrane, a distribution confirmed by cellular fractionation and Western blot analysis. In vitro translation and ex vivo expression showed that RhCG carries a complex N-glycan, probably at the (48)NLS(50) sequon of exoloop 1. These results pinpoint RhCG and Rhcg as novel polytopic membrane glycoproteins that may function as epithelial transporters maintaining normal homeostatic conditions in kidney and testis.

Plant cells in culture secrete a sulfated peptide named phytosulfokine-alpha (PSK-alpha), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable (125)I-labeled PSK-alpha analog, [N(epsilon)-(4-azidosalicyl)Lys(5)]PSK-alpha (AS-PSK-alpha), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-alpha and partially decreased by PSK-alpha analogs possessing moderate binding activities. In contrast, PSK-alpha analogs that have no biological activity showed no competition for (125)I-AS-PSK-alpha binding, confirming the specificity of binding proteins. Analysis of the affinity of (125)I incorporation into the protein by ligand saturation experiments gave apparent K(d) values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of (125)I-AS-PSK-alpha cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of (125)I-AS-PSK-alpha was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-alpha receptors that mediate the biological activities of PSK-alpha.

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(196) linkages and that HN ectodomains dimerize via Cys(123). Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cys(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subsequent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cys(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys(344)-Cys(461) and Cys(455)-Cys(465) linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.

LOX-1 (lectin-like oxidized low density lipoprotein receptor-1) is a type II membrane protein belonging to the C-type lectin family that can act as a cell-surface receptor for atherogenic oxidized low density lipoprotein (Ox-LDL) and may play crucial roles in atherogenesis. In this study, we show, by pulse-chase labeling and glycosidase digestion, that LOX-1 is synthesized as a 40-kDa precursor protein with N-linked high mannose carbohydrate chains (pre-LOX-1), which is subsequently further glycosylated and processed into the 48-kDa mature form within 40 min. Furthermore, when treated with an N-glycosylation inhibitor, tunicamycin, both tumor necrosis factor-alpha-activated bovine aortic endothelial cells and CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) exclusively produced a 32-kDa deglycosylated form of LOX-1. Cell enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence confocal microscopy demonstrated that the deglycosylated form of LOX-1 is not efficiently transported to the cell surface, but is retained in the endoplasmic reticulum or Golgi apparatus in tumor necrosis factor-alpha-activated bovine aortic endothelial cells, but not in BLOX-1-CHO cells. Radiolabeled Ox-LDL binding studies revealed that the deglycosylated form of LOX-1 expressed on the cell surface of BLOX-1-CHO cells has a reduced affinity for Ox-LDL binding. Taken together, N-linked glycosylation appears to play key roles in the cell-surface expression and ligand binding of LOX-1.

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.

Peptide:N-glycanase (PNGase; EC 3.5.1.52) activity was detected in dormant rice seeds (Oryza sativa) and the imbibed rice grains. Time-course studies revealed that the enzyme activity remained almost constant until about 30 h after imbibition in both of endosperm- and embryo tissue-containing areas, and started to increase only in growing germ part, reached a peak at about 3-day stage, followed by a gradual decrease concomitant with a sharp increase in the coleoptile. The specific activity increased about 6-fold at about 3-day stage. PNGase was purified to electrophoretic homogeneity from the extracts of germinated rice seeds at 24 h, and the apparent molecular weight of the purified enzyme, estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), was about 80,000. The purified enzyme was designated PNGase Os to denote its origin. The N-terminal sequence of the 10 residues was determined to be SYNVASVAGL. The purified PNGase Os in SDS-PAGE appeared as a rather broad band, consistent with the presence of multiple glycoforms as indicated by chromatographic behavior on a Sephadex G-75 column. PNGase expressed in coleoptile under anoxia condition was also purified, and both of the purified enzymes were found to exhibit very similar, if not identical, electrophoretic mobility in SDS-PAGE. PNGase Os exhibited a broad pH-activity profile with an optimum of 4-5 and, interestingly, was significantly inactivated by K(+) and Na(+) at near the physiological concentration, 100 mM. These results are discussed in relation to other work.

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/microgram; 76 IU/pmol) was significantly greater than that of Epo (352 IU/microgram; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.

To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

A novel, somewhat basic noncollagenous protein was purified from guanidine hydrochloride extracts of human articular cartilage using cesium chloride density gradient centrifugation, followed by ion-exchange chromatography at pH 5, and gel filtration on two serially coupled columns of Superose 6 and Superdex 200. The protein of 91.5 kDa contains a single polypeptide chain substituted with N-linked oligosaccharides. It appeared unique to cartilage as studied by enzyme-linked immunosorbent assay and immunoblots of various tissue extracts. Its concentration in articular cartilages showed some variability with age being lower in young individuals. It represents a chondrocyte product, since it is synthesized by articular chondrocytes in explant cultures. Interestingly, the distribution of the protein in the articular cartilage provides important information on the nature of chondrocytes at different compartments in the tissue. Thus, chondrocytes in the middle/deeper layers of the tissue in particular, appeared to have produced the protein and deposited it in the interterritorial matrix. The protein was neither seen in the superficial nor in the deepest regions of the articular cartilage. Based on its immunolocalization we have named this protein CILP (cartilage intermediate layer protein).

Several lines of evidence suggest that soluble peptide:N-glycanase (PNGase) is involved in the quality control system for newly synthesized glycoproteins in mammalian cells. Here we report the occurrence of a soluble PNGase activity in Saccharomyces cerevisiae. The enzyme, which was recovered in the cytosolic fraction, has a neutral pH optimum, and dithiothreitol is required for activity. All of these properties were similar to those of earlier described for mammalian PNGases. Interestingly, the yeast enzyme activity was found to be present almost exclusively in cells in stationary phase; little activity was detected in logarithmic growth phase cells. Upon incubation of a glycosylatable peptide R-Asn-X-Thr-R' with permeabilized yeast spheroplasts, we detected formation of both glycosylated peptide and the peptide product expected from PNGase-mediated deglycosylation of this glycopeptide, namely, R-Asp-X-Thr-R'. Recent findings that yeast have an active system for the retrograde transport of unfolded (glyco)proteins and glycopeptides out of the endoplasmic reticulum (ER) into the cytosol raise the possibility that this PNGase may participate in an early step in degradation of these molecules following their export from the ER.

Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact sites between the two organelles. Furthermore, the presence of this glycoprotein in at least two major mitochondrial multienzyme complexes would be consistent with a role in mitochondrial translocations.

d-Tubocurarine (dTC) is a potent competitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds non-equivalently to the two agonist sites (Kd values of 30 nM and 8 microM). When nAChR-rich membranes equilibrated with [3H]dTC are irradiated with 254 nm UV light, [3H]dTC is covalently incorporated into the alpha-, gamma-, and delta-subunits in a concentration-dependent and agonist-inhibitable manner, consistent with the localization of the high and low affinity dTC binding sites at the alpha-gamma- and alpha-delta-subunit interfaces, respectively (Pedersen, S. E. and Cohen, J. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2785-2789). We report on the amino acids within alpha-, gamma-, and delta-subunits that are the sites of specific photoincorporation of [3H]dTC. Subunits isolated from nAChR-rich membranes photolabeled with [3H]dTC were subjected to enzymatic digestion, and peptides containing 3H were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. Isolated peptides were then subjected to NH2-terminal sequence analysis to identify specifically labeled residues. Within the alpha-subunit, 95% of specific incorporation was contained within a 20-kDa proteolytic fragment beginning at Ser-173, with alphaTyr-190 the primary site of [3H]dTC photoincorporation and alphaCys-192 and alphaTyr-198 labeled at lower efficiency. Within gamma- and delta-subunits, specific labeling was contained within proteolytic fragments of 14 and 21 kDa, respectively, beginning at gammaAla-49 and deltaThr-51. gammaTrp-55 and deltaTrp-57 were identified as the sites of specific [3H]dTC photoincorporation. Sequence alignment studies reveal gammaTrp-55 and deltaTrp-57 to be homologous residues at whose position in receptor subunit primary structure a unique pattern of conservation exists in all nAChR (neuronal and muscle). Specifically, all subunits that associate with an alpha-subunit to form an agonist site contain a tryptophan homologous to gammaTrp-55/deltaTrp-57. This pattern of conservation may indicate a functional significance for tryptophan at that location in all nAChR agonist sites.

The G-protein-coupled calcium receptor plays a key role in extracellular calcium homeostasis. To examine the role of the membrane-spanning domains and the approximately 200-residue cytoplasmic carboxyl terminus of the calcium receptor in cell-surface expression and signal transduction, we transfected HEK-293 cells with a series of truncation and carboxyl-terminal missense mutants and analyzed expression by immunoblotting, glycosidase digestion, intact cell immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, respectively, were not properly glycosylated, failed to reach the cell-surface, and showed no calcium response, indicating that mutant receptors with the full extracellular domain but only three or five transmembrane domains are improperly folded and/or processed. Truncation mutants terminating at residues 888 and 903 within the carboxyl terminus were equivalent to the wild type in all assays, whereas mutants truncated at residues 865 and 874 showed no response to calcium, despite only approximately 25% reduction in cell-surface expression. Mutants with a full-length carboxyl terminus but with residues between positions 874 and 888 replaced with alanines showed either no (Ala875, Ala876, and Ala879) or significantly reduced (Ala881-Ala883) calcium response at levels of cell-surface expression equivalent to those of the wild-type receptor. These results indicate that deletion of the majority of the carboxyl terminus is compatible with normal processing, cell-surface expression, and signal transduction of the receptor. The truncation and alanine substitution mutants identify a small region between residues 874 and 888 critical for normal signal transduction by the receptor.

Glycoamidases (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal. While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The Km and Vmax values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 micromol/min/mg, respectively. A natural glycodipeptide, Man9-GlcNAc2-Asn-Phe, was also completely hydrolyzed by glycoamidase A.

Vascular endothelial growth factor (VEGF) is an angiogenesis factor for which two signaling protein tyrosine kinase receptors, Flt1 and KDR, have been identified. We describe here a 190-kDa component present on a human glioma cell line that binds VEGF165 with high affinity. In contrast, VEGF121 is bound only with low affinity, suggesting that the C-terminal part of VEGF165 is important for interaction with the 190-kDa component. No internalization or stimulation of tyrosine phosphorylation was recorded after ligand binding to the 190-kDa component, suggesting that it may not be directly involved in signaling; its function may be to present ligand or stabilize ligand binding to signaling receptors.

The mdm2 oncogene encodes a 90-kDa protein that can bind to the p53 tumor suppressor protein and negatively regulate its functions in transcription, cell cycle arrest, and apoptosis. The mdm2 gene is frequently amplified in human sarcomas, which may be responsible for the malignant transformations. We present evidence that the mdm2 oncoprotein is cleaved by an interleukin 1beta-converting enzyme-like protease (caspase) during p53-mediated apoptosis. The protease that cleaves mdm2 has a specificity similar to that of CPP32 (caspase-3), and recombinant caspase-3 is able to cleave mdm2 in vitro. The protease cleavage site has been mapped to between residue 361 and 362 of human mdm2. The proteolytic cleavage removes the COOH-terminal RING finger domain of mdm2, resulting in the loss of RNA binding activity. The p53 binding and inhibition functions of mdm2 are not affected by the cleavage. The cleavage site sequence of mdm2 is evolutionarily conserved, suggesting that regulation by caspase cleavage during apoptosis is an important feature of mdm2.

O-Linked fucose is an unusual form of glycosylation recently shown to modify the hydroxyls of serine or threonine residues at a strict consensus site within epidermal growth factor-like domains of several serum proteins. Here we demonstrate that Chinese hamster ovary cells modify numerous proteins with O-linked fucose and that the fucose is elongated on specific proteins. We have identified at least two forms of O-linked fucose elongation in Chinese hamster ovary cells: a disaccharide (Glcbeta1,3Fuc) and a larger oligosaccharide of indeterminate structure. Interestingly, it appears that the level of monosaccharide accumulates in the cells over time whereas the disaccharide does not. Analysis of the O-linked fucose-containing saccharides on individual proteins revealed that some proteins are modified with the monosaccharide only, whereas others are modified with monosaccharide and disaccharide, or monosaccharide and oligosaccharide. These results suggest that elongation of the O-linked fucose monosaccharide is a protein-specific phenomena. The presence of elongated O-linked fucose moieties suggests that a novel glycosylation pathway exists in mammalian cells with O-linked fucose as the core.

Anti-peptide antibodies directed against the C-terminal portion (amino acids 603-618) of the rat thyroid iodide transporter (rTIT) have been produced to characterize the molecular forms of rTIT in the rat thyroid and in the functional rat thyroid cell line, FRTL-5. rTIT is located on the basolateral membrane of rat thyroid follicular cells and randomly distributed on the plasma membrane of FRTL-5 cells that do not exhibit cell polarity. The major rTIT component corresponds to an 80-90-kDa glycosylated protein. After treatment of cell membrane fractions with N-glycosidase F or incubation of FRTL-5 cells with tunicamycin, rTIT has an apparent molecular mass of about 55 kDa. FRTL-5 cells cultured in the presence of TSH exhibit a high rTIT content and a high iodide uptake activity (IUA). Upon either removal of TSH or addition of cycloheximide, IUA declines more rapidly than rTIT. The half-life of rTIT was about 4 days. Re-exposure of 7-day TSH-deprived FRTL-5 cells to TSH causes a rapid synthesis of the glycosylated rTIT but a delayed re-induction of IUA. Tunicamycin totally prevents the TSH-dependent re-expression and activity of rTIT. Our data bring basic information on the location, structure, and turnover of rTIT and suggest that its activity is subjected to diverse control mechanisms including regulatory proteins.

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.

A material of Mr 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-alpha-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35S]sulfate and [6-3H]GlcN gave a single component of Mr 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3H/35S material with PNGase F produced two fragments of Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4, 000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 6,000 and 4, 000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [32P]orthophosphate, where radiolabel was incorporated into material of Mr 24,000 and into the fragment of Mr 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3H]His which after chemical deglycosylation produced a major component of Mr 4,000. These results suggest a model for the Mr 24, 000 material consisting of a central polypeptide chain of Mr 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant protein modification found on nuclear and cytoplasmic proteins. Several lines of evidence suggest that it is a highly dynamic modification and that the levels of this sugar on proteins may be regulated. Previous workers (Chou, C. F., and Omary, M. B. (1993) J. Biol. Chem. 268, 4465-4472) have shown that mitotic arrest with microtubule-destabilizing agents such as nocodazole causes an increase in the O-GlcNAc levels on keratins in the human colon cancer cell line HT29. We have sought to determine whether this increase in glycosylation is a general (i.e. occurring on many proteins) or a limited (i.e. occurring only on the keratins) process. A general increase would suggest that the microtubule-destabilizing agents were somehow affecting the enzymes responsible for addition and/or removal of O-GlcNAc. Our results suggest that the changes in O-GlcNAc induced by nocodazole are selective for the keratins. The levels of O-GlcNAc on other proteins, including the nuclear pore protein p62 and the transcription factor Sp1, are not significantly affected by this treatment. In agreement with these findings, nocodazole treatment caused no change in the activity of the enzymes responsible for addition or removal of O-GlcNAc as determined by direct in vitro assay. Interestingly, nocodazole treatment did cause a dramatic increase in modification of N-glycans with terminal GlcNAc residues on numerous proteins. Potential mechanisms for this and the change in glycosylation of the keratins are discussed.

Point mutations in the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor have been shown to cause constitutive activation which results in precocious puberty in affected males. We introduced one of these mutations, Asp-556 --> Gly, into the rat LH/hCG receptor and demonstrated that the mutant receptor constitutively activated adenylate cyclase in transfected 293 T cells. The cell surface expression of the mutant receptor was lower than that of the wild type receptor. Pulse-chase studies showed that the 73-kDa precursor of both the mutant and wild type receptors was synthesized at comparable efficiencies. However, post-translational processing of the mutant receptor to the mature 92-kDa form, which has N-linked complex type oligosaccharide chains, was impaired. Sensitivity of the mutant receptor to peptide-N-glycanase F and endoglycosidase H, and insensitivity to sialidase indicated that the 73-kDa species represents the high mannose form that has not yet been trafficked through the medial and trans Golgi. Additionally, although the wild type receptor was palmitoylated, the mutant receptor was not. Although the high mannose 73-kDa species is capable of binding LH/hCG, our results show that post-translational processing in the Golgi is required for the mature 92-kDa receptor to reach the cell surface.

Aflatoxin B1 is a potent environmental carcinogen produced by certain strains of Aspergillus. Central to the biosynthesis of this mycotoxin is the reaction catalyzed by versicolorin B synthase (VBS) in which a racemic substrate, versiconal hemiacetal, is cyclized to an optically active product whose absolute configuration is crucial to the interaction of aflatoxin B1 with DNA. Attempted over-production of VBS in Escherichia coli led principally to protein aggregated into inclusion bodies but also small amounts of soluble but catalytically inactive enzyme. Comparisons to wild-type VBS by SDS-polyacrylamide gel electrophoresis and after N-glycosidase F treatment revealed that extensive glycosylation accounted for the mass discrepancy (7,000+/-1,500 Da) between the native and bacterially expressed proteins. Several over-expression systems in Saccharomyces cerevisiae were surveyed in which one that incorporated a secretion signal was found most successful. VBS of indistinguishable mass on SDS-polyacrylamide gel electrophoresis and kinetic properties from the wild-type enzyme could be obtained in 50-100-fold greater amounts and whose catalytic behavior has been examined. The translated protein sequence of VBS showed three potential N-glycosylation sites (Asn-Xaa-Ser/Thr) consistent with the modifications observed above and unexpectedly revealed extensive homology to the ADP-binding region prominently conserved in the glucose-methanol-choline (GMC) family of flavoenzymes. Over-production of VBS in yeast marks the first aflatoxin biosynthetic enzyme to be so obtained and opens the way to direct study of the enzymology of this complex biosynthetic pathway.

A theoretical 12-transmembrane segment model based on the hydrophobic moment has been proposed for the transmembrane topology of the glycine transporter GLYT1 and all other members of the sodium- and chloride-dependent transporter family. We tested this model by introducing N-glycosylation sites along the GLYT1 sequence as reporter for an extracellular localization and by an in vitro transcription/translation assay that allows the analysis of the topogenic properties of different segments of the protein. The data reported herein are compatible with the existence of 12 transmembrane segments, but support a rearrangement of the first third of the protein. Contrary to prediction, hydrophobic domain 1 seems not to span the membrane, and the loop connecting hydrophobic domains 2 and 3, formerly believed to be intracellular, appears to be extracellularly located. In agreement with the theoretical model, we provide evidence for the extracellular localization of loops between hydrophobic segments 5 and 6, 7 and 8, 9 and 10, and 11 and 12.

Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N-linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase(R), oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 microM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.

Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.

In this study we demonstrate that the binding region of recombinant truncated human bone osteonectin (tHON) for type V collagen resides between amino acids 1 and 146. After removal of oligosaccharide chain structures from tHON, bovine bone osteonectin (BBON) and human platelet osteonectin (HPON) by N-glycanase, their ability to bind to type V collagen is increased, and HPON affinity to collagen V is the same as that of BBON. These data suggest that glycosylation of osteonectin has a direct or regulatory effect on osteonectin binding to collagen V and that the increase in tHON binding upon removal of carbohydrate is the result of a loss of a down-regulation site or direct interference of the carbohydrate at the binding site. To determine the specific role of each N-glycosylation site in tHON, Asn71 and Asn99 were mutated to Gln (N71Q, N99Q) and Thr73 and Thr101 mutated to Ala (T73A, T101A) to selectively inhibit oligosaccharide attachment. The binding affinity of N99Q and T101Q to collagen V is markedly increased over wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON. The doubled mutant (N71,99Q) binds identically to collagen V as N99Q and T101A. These data suggest that only the position 99 glycosylation site (Asn99-X-Thr101) in tHON is important in the reduction of binding of osteonectin to collagen V. Consistent with the binding data is the observation that both the N71Q and T73A mutant proteins migrate on SDS-polyacrylamide gel electrophoresis gels identically to wild-type tHON, suggesting that there is little or no N-glycosylation of residue 71 in wild-type osteonectin.

Carbohydrate binding to peptide: N-glycanase from mouse fibroblast L-929 cells (L-929 PNGase) and inhibition by oligosaccharides of its catalytic activity were studied. L-929 PNGase was found to bind strongly with oligosaccharides having triomannosido-N,N'-diacetyl-chitobiosyl (Man3GlcNAc2) structure (Kd = approximately 10 microM). This binding was inhibited by mannotriose (Man3; Man alpha 1-->3[Man alpha 1-->6]Man) but not by N,N'-diacetylchitobiose (GlcNAc2; GlcNAc beta 1-->4GlcNAc). Scatchard analysis indicated that there exist two binding sites for Man3 on a homodimeric form of a 105-kDa subunit. Oligosaccharides having Man3GlcNAc2 structure were also shown to be strong inhibitors for the PNGase-catalyzed reaction (Ki = approximately 10 microM). The minimum structural requirements for inhibition of the PNGase activity were Man3 and GlcNAc2. Enzyme kinetic studies showed that the mechanism of inhibition by the oligosaccharides and Man3 fits well with a model wherein two inhibitor binding sites reside on L-929 PNGase. The conformity of Kd with IC50 values may be taken as an evidence for inhibition of the catalytic activity by the oligosaccharides and Man3 through the occupation of the binding sites with these molecules. On the other hand, inhibition by GlcNAc2 followed the simple competitive mode. Since the minimum substrate for the L-929 PNGase was shown to be Man beta 1-->4GlcNAc beta 1-->4GlcNAc beta 1-->peptide, GlcNAc2 may be directly accessible to the catalytic site in competition with substrate. Interestingly, alkylation of -SH group in L-929 PNGase caused complete loss of the catalytic activity, but the carbohydrate binding activity was completely retained, indicating that the catalytic site(s) is discriminated from the carbohydrate-binding sites in the active site of this enzyme. The carbohydrate-binding property seems to be unique to soluble PNGases from mammals and may be associated not only with regulation of the enzyme activity, but also with receptor and carrier functions for glycoconjugates in certain intracellular processes.

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.

The glycoprotein (GP) Ib-IX complex is the receptor on platelet surfaces that mediates their adhesion to subendothelium. It comprises three polypeptides (GP Ib alpha, GP Ib beta, GP IX), each of which belongs to a superfamily of proteins containing conserved leucine-rich motifs. In this study, we used Chinese hamster ovary (CHO) cells expressing every combination of two GP Ib-IX complex subunits to demonstrate that GP Ib beta plays an essential role in the synthesis of the heterotrimer by associating with both of the other two subunits. Confocal microscopy demonstrated that GP Ib beta was present in the same cellular locations as GP Ib alpha in CHO alpha beta cells (cells expressing only GP Ib alpha and GP Ib beta) and as GP IX in CHO beta IX cells. The two polypeptides expressed in CHO alpha IX cells did not co-localize. Association between GP Ib alpha and GP Ib beta was demonstrated biochemically on immunoblots of detergent lysates of CHO alpha beta cells; electrophoresis under nonreducing conditions revealed the two subunits to be covalently linked through a disulfide bond. Association of GP Ib alpha and GP Ib beta was further demonstrated by the finding that immunoprecipitations with antibodies against either polypeptide precipitated both. Similarly, immunoprecipitations of lysates of CHO beta IX cells with antibodies against GP Ib beta or GP IX precipitated both polypeptides. In contrast, co-immunoprecipitation of the two polypeptides expressed in CHO alpha IX cells could not be demonstrated. Transient expression in CHO cells of GP Ib beta with GP IX yielded higher GP IX levels on the cell membrane than did expression of GP IX alone; supertransfection of CHO alpha IX cells with GP Ib beta also increased GP IX levels on the cell surface.

Recently, we found the occurrence of N-deglycosylating enzyme, peptide:N-glycanase (PNGase), in mammalian cells and observed that PNGase is a rather common enzyme involved in post-translational remodification of proteins (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1993) Biochem. Biophys. Res. Commun. 194, 1124-1130). We report here a 460-fold purification to homogeneity with 11.5% yield of PNGase from crude extract of C3H mouse-derived L-929 fibroblast cells. The purified enzyme, designated as L-929 PNGase, had the apparent molecular weight of 212,000 and was composed of two 105,000 subunits. Although this enzyme was capable of hydrolyzing structurally diverse natural glycopeptide substrates bearing high mannose, hybrid, and complex-type glycan units, the activity was completely inhibited by the presence of the fucose residue either alpha-1-->3- or alpha-1-->6-linked to the proximal GlcNAc residue. The enzyme showed maximal activity at pH near 7. This and the inability to act on glycoasparagine strongly support our view that this enzyme would not be involved in lysosomal degradation pathway. L-929 PNGase was characterized by having distinctly a low Km value, which may be of physiological significance. Possible wide occurrence of N-deglycosylation of glycoproteins was shown by a data bank survey of the protein sequences showing discrepancies between those determined directly (-D-X-(S/T)-) and those deduced from cDNA sequencing (-N-X-(S/T)-). We propose here that PNGase-catalyzed N-deglycosylation is a functionally important universal feature in living cells.