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We tested the importance of the aspartate-any residue-aspartate (DXD) motif for the enzymatic activity and nucleotide binding capacity of the Golgi glycosyltransferase GM2 synthase. We prepared point mutations of the motif, which is found in the sequence 352-VLWVDDDFV, and analyzed cells that stably expressed the mutated proteins. Whereas the folding of the mutated proteins was not seriously disrupted as judged by assembly into homodimers, Golgi localization, and secretion of a soluble form of the enzyme, exchange of the highly conserved aspartic acid residues at position 356 or 358 with alanine or asparagine reduced enzyme activity to background levels. In contrast, the D356E and D357N mutations retained weak activity, while the activity of V352A and W354A mutants was 167% and 24% that of wild-type enzyme, respectively. Despite the major effect of the DXD motif on enzymatic activity, nucleotide binding was not altered in the triple mutant D356N/D357N/D358N as revealed by binding to UDP-beads and labeling with the photoaffinity reagent, P(3)-(4-azidoanilido)uridine 5'-triphosphate (AAUTP). In summary, rather than being critical for nucleotide binding, this motif may function during catalysis in GM2 synthase, as has been proposed elsewhere for the SpsA glycosyltransferase based on its crystal structure.

Ganglioside GM2 synthase and other enzymes required for complex ganglioside synthesis were localized recently to the trans Golgi network (TGN). However, there are conflicting reports as to the location of GM3 synthase; originally this enzyme was detected in the early Golgi of rat liver but a recent report localized it to the late Golgi. We have used chimeric forms of ganglioside GM2 synthase to determine if the location of lactosylceramide (LacCer) to GM3 conversion in Chinese hamster ovary (CHO) cells was the early or late Golgi. Our approach tested whether GM3 could be utilized as a substrate by GM2 synthase chimeras which were targeted to compartments earlier than the trans Golgi, i.e., GM3 produced in the cis Golgi should be utilized by GM2 synthase located anywhere in the Golgi whereas GM3 produced in the trans Golgi should only be used by GM2 synthase located in the trans Golgi or TGN. Comparison of cell lines stably expressing these chimeras revealed that the in vivo functional activity of GM2 synthase decreased progressively as the enzyme was targeted to earlier compartments; specifically, the percentage of GM3 converted to GM2 was 83-86% for wild type enzyme, 70% for the medial Golgi targeted enzyme, 13% for the ER and cis Golgi targeted enzyme, and only 1.7% for the ER targeted enzyme. Thus, these data are consistent with a late Golgi location for LacCer to GM3 conversion in these cells.

Brefeldin A reversibly disassembles the Golgi complex, causing mixing of the Golgi cisternae with the ER while the trans Golgi network persists as part of a separate endosomal membrane system. Because of this compartmental separation, Brefeldin A treatment has been used to map the sub-Golgi locations of several Golgi enzymes including GM2 synthase. We previously proposed that GM2 synthase might be located in a distal portion of the Golgi complex which in the presence of Brefeldin A would be separated from the substrate ganglioside GM3 present in the mixed ER-Golgi membrane system. In the present study we show using GM2 synthase chimeras that GM2 synthesis was blocked by Brefeldin A when GM2 synthase was distributed throughout all Golgi subcompartments or even when it was restricted to the medial Golgi. Because these findings opposed our speculation regarding a distal location of this enzyme, we sought an alternative explanation for the inhibition of ganglioside synthesis by Brefeldin A. However, Brefeldin A did not degrade GM2 synthase, prevent its homodimerization, or inhibit its in vitro activity. Brefeldin A did result in the conversion of a portion of membrane bound GM2 synthase into a soluble form which has minimal capability to produce GM2 in whole cells. However, this conversion was not sufficient to explain the nearly total loss of GM2 production in intact cells in the presence of Brefeldin A. Nevertheless, the results of this study indicate that Brefeldin A-induced inhibition of ganglioside synthesis cannot be used to deduce the location of GM2 synthase.

A wide-area network (WAN) connecting a new drug informatics center in a university-affiliated hospital with the university's campus-based computer network is described. In 1994 a pharmacy school developed a drug informatics center in an affiliated hospital. The center was originally designed around a local-area network (LAN) to be located at the hospital and planned to provide clients with easy access to typical productivity software and various electronic information resources. Only occasional modem connections to the university network were envisioned. However, large price increases in information retrieval systems and decreases in the cost of a frame relay connection (T1 line) to the campus network led to the installation of a WAN when the drug informatics center was established. Technical, political, and legal problems were overcome, and the connection was made. The WAN gave faculty and students at the hospital access to many of the university's computing and Internet resources. In addition, the faculty and students have access to various files and programs available only on the drug informatics center's file server at the affiliated hospital. It cost about $6500 to install all WAN equipment and maintain the frame relay for the first year, or a third of what would have been necessary for information retrieval software had a separate LAN been established at the hospital. A WAN connecting a drug informatics center and a university's computer network gave the center access to more electronic information resources at lower cost than would have been possible with a separate LAN.

High-gradient magnetic affinity chromatography (HIMAC) has been used to obtain highly enriched plasma membranes, free of intracellular membrane contaminants, from cultured Chinese hamster ovary (CHO) cells in yields of > or = 80%. Using this procedure we have characterized the transport of glucosylceramide (GlcCer) and the ganglioside GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sphingomyelin to the plasma membrane but has no effect on the transport of GlcCer. Similarly, incubation of CHO cells at 15 degrees C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GlcCer movement. We propose that carrier-mediated transport accounts for a major fraction of the plasma membrane GlcCer. Pulse-chase studies with either [3H]glucose or [3H]palmitate indicate that newly synthesized GlcCer which has reached the plasma membrane is not utilized for the synthesis of GM3 but is instead rapidly either degraded or converted into an as yet unidentified product. Our results indicate that in addition to serving as a precursor for higher glycosylation in the Golgi, a major fraction of newly synthesized GlcCer is rapidly transported to the plasma membrane by a non-Golgi pathway and then rapidly turned over.

Mice challenged with L5178Y lymphoma cells expression high levels of the glycolipid asialo GM2 (gangliotriosylceramide) were protected from tumor growth by passive administration of a monoclonal antibody specific for the glycolipid; in a few antibody-treated mice, ascites cells eventually proliferated which contained a reduced chemical quantity of the glycolipid antigen (3). We now report that the cells emerging from antibody-treated mice had abnormal marker chromosomes identical to those in the cells used for challenge, indicating that the emergent cells were progeny of the challenge inoculum. Flow cytometric analysis revealed that asialo GM2 was undetectable on the surface of greater than 95% of the tumor cells from antibody-treated mice, whereas surface display of H-2 determinants was unchanged from that of the cells used for challenge. Tumor cells arising in challenged but untreated mice consisted of a mixture of asialo GM2-positive and -negative cells, indicating the presence of selective pressures in these mice as well. None of the cells taken from tumor bearing mice differed significantly from the challenge cells in their susceptibility to natural killer cell attack, suggesting that resistance to natural killer cell lysis was not responsible for the proliferation of these cells in vivo. When cells derived from an antibody-treated mouse were used to challenge mice, serotherapy with anti-asialo GM2 had no effect on mouse survival. These results suggest that serotherapy may complement a host anti-tumor response, from which only asialo GM2 deficient cells can escape.

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.

A simplified method is presented for the oxidation of the olefinic bond of the sphingosine moiety of glycosphingolipids to a carboxyl group. Coupling of such “glycolipid acids” to glass beads, agarose gels, proteins, and polyacrylic hydrazide polymers is described. Solid supports and macromolecules that have been derivatized in this fashion are useful reagents for a variety of studies in cell biology and immunology.

Many Golgi membrane-bound glycosyltransferases are released from cells in a soluble form. To characterize this release process, we stably transfected Chinese hamster ovary cells with three myc epitope-tagged forms of cloned beta1, 4-N-acetylgalactosaminyltransferase (GalNAcT); two of these forms resided in the Golgi, while the third was retained in the ER. GalNAcT was released into the culture medium from cells transfected with the Golgi forms but not with the ER form of the enzyme. The medium from cells transfected with the Golgi forms contained disulfide-bonded dimers of GalNAcT, which carried neuraminidase sensitive, complex N-linked carbohydrate chains. This soluble species represented the major degradation product of cellular GalNAcT, which turned over with a half-time of about 1.7 h. The soluble species consisted of a mixture of truncated GalNAcT molecules, the major form of which was produced by cleavage near the boundary between the transmembrane and lumenal domains between Leu-23 and Tyr-24. This cleavage site fits the sequence pattern for sites cleaved by cathepsin D (van Noort, J.M., and van der Drift, A. C.M. (1989) J. Biol. Chem. 264, 14159-14164). These findings suggest that GalNAcT is converted from a membrane-bound to a soluble form as a result of cleavage by a cathepsin D-like protease in a compartment late in the Golgi secretory pathway.

Two hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of BALB/c mice that had been immunized with the glycolipid ganglio-N-triosylceramide (asialo GM2). The specificity of the monoclonal antibodies produced by these hybridomas, one an IgM and the other an IgG3, has been defined by hemagglutination inhibition, complement fixation, and lysis of glycolipid liposomes by antibody and complement. A major determinant recognized by the IgM antibody is the nonreducing terminal N-acetylgalactosamine including the C6 primary hydroxyl group, but excluding the C2-acetamide group of N-acetylgalactosamine, because oxidation with galactose oxidase produced a structure showing only minimal cross-reaction with the IgM but replacement of the N-acetyl group with an N-n-butyryl group produced a glycolipid that reacts with IgM antibody to the same extent as with the unmodified glycoplipd. A major determinant recognized by the IgG3 antibody is the terminal N-acetylgalactosamine including the C2-acetamido group, but excluding the C6 primary hydroxyl group of N-acetylgalactosamine, because replacement of the N-acetyl group with an N-n-butyryl group produced a glycolipid that did not react with the IgG3 antibody; in striking contrast the IgG3 antibody reacted with the C6-oxidized glycolipid as well as with the native glycolipid. Neither antibody reacted significantly with any other natural glycolipids tested including several that are structurally related to asialo GM2 such as ganglioside GM2, ganglio-N-tetraosylceramide (asialo GM1), or ceramide dihexoside. These results indicated that in addition to the fine structure specificity described above both antibodies recognize the nonreducing terminal GalNAc beta 1 leads to 4Gal structure. The strict antigenic specificity of these monoclonal anti-glycolipid antibodies indicates their great potential as specific probes for cell surface studies.

The pattern of distribution of antigens cross-reacting with antibodies to human blood group antigens A and B and two precursor molecules was examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice. Staining for blood group antigen A was negative. In all epithelia examined, blood group antigen B was present at the surface of basal and parabasal cells, and the H antigen at the surface of spinous cells. N-acetyllactosamine was present on the cell membranes in the upper spinous and granular cell layers of epidermis and forestomach epithelium and was not expressed in the oral epithelia except for a limited area in the dorsal tongue epithelium.

Brefeldin A (BFA) induces the rapid redistribution of the Golgi complex into the endoplasmic reticulum (ER), causing the glycoproteins that are retained in the ER to be processed by Golgi enzymes. We have examined the effects of BFA on the synthesis of glycosphingolipids (GSL) to map the intracellular sites of GSL synthesis. In several cultured cell types, BFA inhibited the synthesis of the neutral GSL gangliotriaosylceramide (GA2) and monosialoganglioside GM2 and disialoganglioside GD2, where GD2 is GalNAc(beta 1----4)- [NeuAc(alpha 2----8)NeuAc(alpha 2----3)]Gal(beta 1----4)GlcCer, GM2 lacks the NeuAc(alpha 2----8) unit, and GA2 lacks both NeuAc(alpha 2----8) and NeuAc(alpha 2----3) units. The observed decrease in labeling of GA2, GM2, and GD2 in the presence of BFA was not due either to enhanced degradation of these glycolipids or to shedding of these glycolipids from the cells. In rat liver all three of these glycolipids have been shown by others to be synthesized by the same enzyme, GA2/GM2/GD2 synthase, which catalyzes the addition of N-acetylgalactosamine to lactosylceramide (Lac-Cer), GM3 [NeuAc(alpha 2----3)Gal(beta 1----4)GlcCer], and GD3 [NeuAc(alpha 2----8)NeuAc-(alpha 2----3)Gal(beta 1----4)GlcCer], respectively. Studies with a fluorescent glycolipid analog indicated that BFA redistributed the trans-Golgi stacks into a reticular pattern characteristic of the ER. These studies localize GA2/GM2/GD2 synthase, a key enzyme involved in the synthesis of complex gangliosides, to a compartment late in the intracellular trafficking pathway, which remains functionally distinct from the ER in the presence of BFA.

Adenylate cyclase (AC) toxins produced by Bacillus anthracisand Bordetella pertussiswere compared for their ability to interact with and intoxicate Chinese hamster ovary cells. At 30 °C, anthrax AC toxin exhibited a lag of 10 min for measurable cAMP accumulation that was not seen with pertussis AC toxin. This finding is consistent with previous data showing inhibition of anthrax AC toxin but not pertussis AC toxin entry by inhibitors of receptor-mediated endocytosis (Gordon, V. M., Leppla, S. H., and Hewlett, E. L. (1988) Infect. Immun. 56, 1066–1069).

Summary Mouse hybridomas were obtained that secrete monoclonal antibodies recognizing glycolipid antigens located in the flagellar membrane of the biflagellate alga,Chlamydomonas reinhardtii. The antigen is an acidic lipid that migrates slightly slower than a GM1 ganglioside on thin layer chromotography. The binding of the antibodies to the thin layer plate was inhibited by periodate oxidation suggesting that the antibodies are recognizing a carbohydrate epitope. In a variety ofChlamydomonas strains, these antibodies were found to stain the flagella of only a sub-set of the cells in the population, generally varying from 50% to 75% of the cells. Even after cloning, the population of cells continued to express this variability in staining, and presumably, expression of the glycolipid epitope. Although most cells showed either strong staining of both flagella or no detectable staining of both flagella, a subset of the cells in the culture exhibited differential antibody labeling of the two flagella, suggesting that an individualChlamydomonas can exhibit a different glycolipid composition in each of its two flagellar membranes and even differential expression along the length of an individual flagellum.

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3-galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.

The heterogenetic Forssman antigen is a glycosphingolipid, a ceramide pentasaccharide with the structure GalNAcα1→3GalNAcβ1→3Galα1→4Galβ1→4Glc→ceramide. Forssman-positive animals are capable of synthesizing this compound in tissues or in erythrocytes, in contrast to the Forssman-negative species, including humans, which are incapable of adding the last carbohydrate in the sequence of the Forssman antigen, namely αGalNAc. The Forssman glycolipid and its precursor globoside were examined in twenty-one samples of surgically extirpated gastrointestinal mucosa and tumors derived therefrom. The results revealed that a few patients had chemically and immunologically detectable levels of the Forssman glycolipid as a normal component of their gastrointestinal mucosa (F+population); in contrast, the majority of patients did not contain this glycolipid in their normal mucosa (F-population). Whereas the F-population included blood groups A, B, and O, the F+population did not correspond to blood group A. The Forssman status in tumors taken from the F+or F-population showed the following striking features: (i) all tumors derived from F-mucosa possessed Forssman glycolipid, whereas (ii) none of the tumors originating in F+mucosa contained Forssman glycolipid. Globoside, the immediate precursor of Forssman antigen, was distributed equally among F+and F-mucosa and the tumors derived therefrom. Thus, the expression of Forssman antigen in gastrointestinal mucosa appears akin to that of an isoantigen. Furthermore, the Forssman antigen that appears in tumors of the F-population could represent a human tumor-associated antigen. In view of the strong crossreactivity of Forssman antigen with blood group A determinants, the appearance of Forssman antigen in human tumors could be related to the “A-like antigen” (or “neo-A antigen”) of human tumors reported previously [Hakomori, S., Koscielak, J., Black, K. J. & Jeanloz, R. W. (1967) J. Immunol. 98, 31-38; Häkkinen, I. (1970) J. Natl. Cancer Inst. 44, 1183-1193].

To characterize the process by which glycolipids are shed from cell membranes, the cellular and supernatant glycolipids were compared from a variant of the mouse lymphoma L5178Y which had been selected for strong expression of the neutral glycolipid gangliotriaosylceramide (GgOse3Cer). This glycolipid was present in three forms which differed in their fatty acid composition. Whereas the major cell-associated form of GgOse3Cer contained C24 fatty acids, the predominant form shed into the culture supernatant contained C16 fatty acids. Ultracentrifugation of the culture medium yielded a pellet with a GgOse3Cer profile similar to that of the cells and a supernatant enriched in the C16 fatty acid form. Gel filtration of the culture medium revealed two GgOse3Cer-containing pools. The first was excluded from Sepharose CL-2B and had a GgOse3Cer profile similar to that of the cells, while the second migrated with proteins in the range of 25,000-500,000 daltons and was enriched in the C16 fatty acid form. These results suggest two forms in which glycolipids are released from cell membranes. The first is in a large complex, possibly a membrane vesicle, which retains the glycolipid profile of the membrane of intact cells while the second form appears to result from the preferential release of particular glycolipid components.

HISTORY. —J., a man aged 45, Swedish, a sheet-metal worker, entered the Peter Bent Brigham Hospital, Sept. 11, 1913, complaining of chills, fever and bloody urine. The family history was unimportant as related to the present illness. The patient had interstitial keratitis at 3 years (?), and measles and small-pox as a child, but no other infectious diseases in childhood. The patient came to the United States nineteen years ago and served in the Navy during the Spanish-American War. He has been a sailor for several years since, usually serving as engineer on a private yacht. Patient has not been south of New York for twelve years. Ten years ago he had an attack of pneumonia; there was no residue. Four years ago in September, patient had a sudden chill, followed in one hour by fever and sweating. Following this attack, he passed dark urine for from six to eight hours.