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Human carboxylesterase 2 (hCES2) is a glycoprotein involved in the metabolism of drugs and several environmental xenobiotics, whose crystallization has been proved to be a challenging task. This limitation could partly be due to glycosylation heterogeneity and has delayed the disclosure of the 3D structure of hCES2 which would be of upmost relevance for the development of new substrates and inhibitors. The present work evaluated the involvement of glycans in hCES2 activity and thermo stability in an attempt to find alternative active forms of the enzyme that might be adequate for structure elucidation. Partial or non-glycosylated forms of a secreted form of hCES2 have been obtained by three approaches: (i) enzymatic deglycosylation with peptide N-glycosidase F; (ii) incubation with the inhibitor tunicamycin; ii) site directed mutagenesis of each or both N-glycosylation sites. Deglycosylated protein did not show a detectable decrease in enzyme activity. On the other hand, tunicamycin led to decreased levels of secreted hCES2 but the enzyme was still active. In agreement, mutation of each and both N-glycosylation sites led to decreased levels of secreted active hCES2. However, the thermostability of the glycosylation mutants was decreased. The results indicated that glycans are involved, to some extent in protein folding in vivo, however, removal of glycans does not abrogate the activity of secreted hCES2., Highlights • Partial or non-glycosylated forms of Human carboxylesterase 2 (hCES2) were produced. • N-glycosylation is not essential for hCES2 activity. • Partial or non-glycosylated forms are less thermostable than the glycosylated form.

Background Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. Methods The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. Results and conclusions One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis x motif ( FUT9 ), polysialic acid ( ST8SIA2 and ST8SIA4 ) and HNK-1 ( B3GAT2 ). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis x structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis x , as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. General Significance These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis x motif plays a functional role in neurite outgrowth from human neurons.

L1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1–4 interacted homophilically in trans, contrary to mutants L1/Ig1–3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 ± 2 and 130 ± 6 nm for L1/ECD-L1/ECD and L1/ECD-L1/Ig1–4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1–4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1–4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1–Ig4 in a horseshoe conformation.

A disintegrin and metalloprotease 10 (ADAM10) is a type I transmembrane glycoprotein with four potential N-glycosylation sites (N267, N278, N439 and N551), that cleaves several plasma membrane proteins. In this work, ADAM10 was found to contain high-mannose and complex-type glycans. Individual N-glycosylation site mutants S269A, T280A, S441A, T553A were constructed, and results indicated that all sites were occupied. T280A was found to accumulate in the endoplasmic reticulum as the non-processed precursor of the enzyme. Furthermore, it exhibited only residual levels of metalloprotease activity in vivo towards the L1 cell adhesion molecule, as well as in vitro, using a ProTNF-alpha peptide as substrate. S441A showed increased ADAM10 susceptibility to proteolysis. Mutation of N267, N439 and N551 did not completely abolish enzyme activity, however, reduced levels were found. ADAM10 is sorted into secretory vesicles, the exosomes. Here, a fraction of ADAM10 from exosomes was found to contain more processed N-linked glycans than the cellular enzyme. In conclusion, N-glycosylation is crucial for ADAM10 processing and resistance to proteolysis, and results suggest that it is required for full-enzyme activity.

A familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), is caused by dominant mutations in the cytosolic Cu,Zn superoxide dismutase (SOD1). There has been evidence for secretion of SOD1, by an unknown mechanism. In this work stable mouse motor neuron-like NSC-34 cells overexpressing human SOD1 wild-type hSOD1(wt) (NSC-34/hSOD1(wt)) and mutant hSOD1(G93A) (NSC-34/hSOD1(G93A)) have been used as an ALS cell model. SOD1 was found to be secreted in association with a membrane fraction that pelleted at 100,000xg. Sucrose density gradient separation of this fraction showed that wild-type and mutant SOD1 were found between 0.5 and 1.16M sucrose and co-localized with the exosomal marker CD9. Therefore, SOD1 secretion occurred via exosomes. p115 a cytosolic and Golgi apparatus (GA) protein involved in vesicle tethering was also found in exosomes, contrary to the endoplasmic reticulum protein calnexin. SOD1 secretion mediated by exosomes could explain cell-to-cell transfer of mutant toxicity.

The transmembrane domain (TM) and flanking regions of glycosyltransferases (GTs) have been implicated in the localization of these proteins in the Golgi apparatus (GA). alpha3/4 Fucosyltransferase III (FT3wt) (EC 2.4.1.65) is localized in the trans-Golgi and trans-Golgi network (TGN) of baby hamster kidney (BHK) cells and synthesizes Lewis determinants associated with cell adhesion events. We have evaluated the effect of removing the cytosolic domain on the localization of the enzyme and its capacity for synthesizing the Lewis A (Le A) determinant. The mutant where the cytoplasmic domain (Asp-2 to Trp-13) of FT3wt has been deleted (FT3dc) was localized in the Golgi but it was shifted to earlier compartments than FT3wt. The mutant was not detected on the plasma membrane (PM) and glycosylation analysis indicated that FT3dc was transported beyond the endoplasmic reticulum (ER) since complex type glycosylation was observed. Cells expressing FT3dc showed a significantly lower efficiency to synthesize Le A when compared with cells expressing FT3wt, in vivo. This reduction was not due to lower specific activity because both enzyme forms had a similar specific activity in vitro. Therefore, removal of FT3 cytosolic tail caused a shift in enzyme distribution to earlier Golgi compartments concomitant to the decrease of its biosynthetic capacity.

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the gamma-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other gamma-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the gamma-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfide-bonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human gamma-secretase components, all of the ceNCT/hNCT chimeras interacted with gamma-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the gamma-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.

Human fucosyltransferase III (EC 2.4.1.65) (FT3wt) is localized in the Golgi of baby hamster kidney cells and synthesizes Lewis determinants associated with cell adhesion events. Replacement of the amino acid residues from the transmembrane domain (TM) Cys-16, Gln-23, Cys-29, and Tyr-33 by Leu (FT3np) caused a shift in enzyme localization to the plasma membrane. The mislocalization caused a dramatic decrease in the amount of biosynthetic products of FT3wt, the Lewis determinants. Determination of the expression levels on the surface with mutants of the enzyme, where one, two, or three of these residues were replaced by Leu, suggested that Cys from the TM was required for the localization of FT3 in the Golgi. Furthermore, Cys-23 and Cys-29 mediated the formation of disulfide-bonded dimers but not higher molecular weight oligomers.In vitro reconstitution of intra-Golgi transport showed that FT3wt was incorporated into coatomer protein (COP) I vesicles, contrary to FT3np. These data suggested that Cys, Gln, and Tyr residues are important for FT3wt sorting into the transport vesicles possibly due to interactions with other membrane proteins.

Human alpha3/4fucosyltransferase (FT3) catalyses the synthesis of fucosylated glycoconjugates involved in cell-cell interactions. FT3 has two potential N-glycosylation sites at Asn(154) and Asn(185). Soluble secretory forms of the enzyme (SFT3) and mutant forms with the first, second and both glycosylation sites (SFT3DN1, SFT3DN2, SFT3DN) mutated have been expressed in baby hamster kidney (BHK) and Spodoptera frugiperda (Sf9) cells. Deletion of the first or both sites caused total enzyme inactivation. Deletion of the second site caused 99% and 75% decrease of secretory enzyme expression in BHK and Sf9 cells, respectively. Sf9 cells produced 1 mg/l SFT3 and 0.3 mg/l SFT3DN2; these values were 175- and 3750-fold higher, respectively, than those observed for BHK cells. A significant amount of protein was accumulated intracellularly in Sf9 cells which for SFT3 was active and for SFT3DN2 was inactive, indicating the importance of the glycans from the second glycosylation site for protein folding. The corresponding full-length forms FT3, FT3DN1 and FT3DN2 associated with calnexin as observed by immunoprecipitation studies, which indicated the possible role of this chaperon in the folding of glycosylated glycosyltransferases.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manβ4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two β-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

Stable BHK-21 cell lines were constructed expressing: (1) the wild-type form of human α1,3/4-fucosyltransferase (FT3T2); and (2) the secretory form of the enzyme, where amino acid residues 46-361 (S2FT3T2) were coupled at their amino terminus to the signal sequence of interleukin-2. Preliminary results of immunofluorescence microscopy indicated that S2FT3T2 was present in the endoplasmic reticulum and Golgi apparatus of the cells, whereas FT3T2 was localized in the Golgi apparatus. The S2FT3T2 was purified by affinity on a GDP-Fractogel resin and its specificity towards oligosaccharides and glycoproteins was studied by HPAEC-PAD, methylation analysis, MALDI/TOF-MS and ESI/MS-MS. The soluble form of α1,3/4-fucosyltransferase may be used for the in vitro synthesis of the Lewis a determinant on carbohydrates and glycoproteins.

Each of the five human alpha1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human beta-trace protein (beta-TP) as a secretory reporter glycoprotein. In order to study their in vivo properties for the transfer of peripheral Fuc onto N-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All fucosyltransferases were found to peripherally fucosylate 19-52% of the diantennary beta-TP N-glycans, and all enzymes were capable of synthesizing the sialyl LewisX (sLex) motif. However, each enzyme produced its own characteristic ratio of sLex/Lex antennae as follows: FT7 (only sLex), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4 (1:7). Fucose transfer onto beta-TP N-glycans was low in FT3 cells (11% of total antennae), whereas the values for FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%, respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Lex on asialo diantennary N-glycans and mono-Lex with monosialo chains. In contrast, FT6 forms mostly alpha1,3-difucosylated chains with no, one, or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium in significant amounts, whereas FT7 and FT5 were found to be largely resistant toward proteolysis. Studies on engineered soluble variants of FT6 indicate that these forms do not significantly contribute to the in vivo fucose transfer activity of the enzyme when expressed at activity levels comparable to those obtained for the wild-type Golgi form of FT6 in the recombinant host cells.

Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human α1,3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galβ1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galβ1-3GlcNAc. The type II motif Galβ1-4GlcNAc in bi-, tri-, or tetraantennary neutral or α2-3/α2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.

The effects of tunicamycin, an inhibitor of lipid-linked glycosyl transfer, and swainsonine, an inhibitor of Golgi mannosidase II, on Lupinus albus L. root growth in vitro were studied. SDS-PAGE analysis of total and endomembrane fraction Con A-binding polypeptides from cultured roots indicated that: (i) tunicamycin effectively prevents N -glycosylation of root polypeptides; (ii) swainsonine inhibits Golgi mannosidase II which results in the formation of N-linked oligosaccharides with extra mannose residues. Increases in root length of 26% and 20% were observed when roots were incubated with 5 and 10 μg/ml tunicamycin, respectively, during 48 h; after this period their growth rate was inhibited. Swainsonine also had a stimulating effect on L. albud root growth during the initial 48 h, for each of the concentrations tested (5, 10 and 15 μg/ml) showing similar effects on growth (30%, 25% and 32%, respectively). During the following 48-h period (up to 96 h) a sustained stimulating effect was observed with 5 and 10 μm/ml swainsonine (29%), while the 15-μg/ml dose exerted a much higher stimulation (46%).