Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

The effect of a "bisecting" N-acetylglucosaminyl group on the binding of biantennary, complex oligosaccharides to concanavalin A, Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin (RCA-120) immobilized on agarose

The effect of a "bisecting" 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, linked (1----4) to the beta-D-mannopyranosyl group of asparagine-linked complex and hybrid oligosaccharides, on the binding of [14C]acetylated glycopeptides to columns of immobilized concanavalin A (Con A), Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin-120 (RCA-120) was investigated. The presence of this "bisecting" GlcNAc group caused significant inhibition of the binding to ConA-agarose of biantennary complex glycopeptides in which the two branches are terminated at their nonreducing ends by two GlcNAc groups, or by a Gal and a GlcNAc group, or by two Gal groups, or by a Man and a GlcNAc group. Binding of biantennary, complex glycopeptides to E-PHA-agarose required a "bisecting" GlcNAc group, a Gal group at the nonreducing terminus of the alpha-D-Man-p-(1----6) branch, and a terminal or internal GlcNAc residue linked beta-(1----2) to the alpha-D-Manp-(1----3) branch. Binding to RCA-120-agarose occurred only when at least one nonreducing terminal Gal group was present, and increased as the proportion of terminal Gal groups increased; the presence of a "bisecting" GlcNAc group caused either enhancement or inhibition of these binding patterns. It is concluded that a "bisecting" GlcNAc group affects the binding of glycopeptides to all three lectin columns.     

Inhibition of lymphocyte 5'-nucleotidase by lectins: effects of lectin specificity and cross-linking ability

5'-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5'-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5'-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5'-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5'-nucleotidase is concanavalin A.     

CHARACTERIZATION AND TOPOGRAPHY OF THE GLYCOPROTEINS OF ADRENAL CHROMAFFIN GRANULES

The glycoproteins of the membranes of bovine chromaffin granules were characterized by two polyacrylamide gel electrophoresis systems. Five components (I-V) were demonstrated with apparent molecular weights ranging in the unreduced form from 45,000 to 150,000. Glycoprotein I was identified as the enzyme dopamine β-hydroxylase. Four of these glycoproteins (with the exception of component IV) were apparently also present in the membranes of pig and horse chromaffin granules. The soluble proteins of chromaffin granules contained at least three glycoproteins. Only glycoprotein I (dopamine β-hydroxylase) was present both in the soluble content and in the membranes of chromaffin granules. Affinity chromatography with lectins demonstrated that from the soluble proteins only dopamine β-hydroxylase was adsorbed by concanavalin A, whereas none of these proteins reacted with wheat germ lectin and Ricinus communis agglutinin. Three membrane proteins including dopamine β-hydroxylase and glycoprotein II as major components were adsorbed by concanavalin A, whereas wheat germ lectin bound only component II and a small amount of component III. By electron microscopy it was demonstrated that concanavalin A did not bind to intact chromaffin granules whereas ruthenium red and cationized ferritin did. Isotope labelling after galactose oxidase treatment revealed that at least the carbohydrate portion of the major glycoproteins is present on the inner side of the granule membranes facing the content.     

Microglial cells in the central nervous system of the rabbit and rat: cytochemical identification using two different lectins

Microglial cells were selectively demonstrated in the central nervous system of adult rabbits and rats using lectin histochemistry. Biotinylated Ricinus communis agglutinin-120 (RCA-1) and biotinylated Griffonia simplicifolia B4 isolectin (GSA I-B4) were used as histochemical markers on sections of Bouin-fixed paraffin-embedded cerebrum and cerebellum. Results were quite similar using both lectins and both species. GSA I-B4 resulted in a better staining in the rat, while RCA-1 labelling was superior in the rabbit. Neither neurons nor glial cells other than microglia were stained with our technique. Lectin histochemistry applied for the detection of microglial cells appears to be of sufficient selectivity and may be considered as an important tool in the morphological and neurobiological study of these cells.     

Studies of the molecular recognition of synthetic methyl beta-lactoside analogues by Ricinus communis agglutinin.

The 2-, 3-, 6-, 2'-, 3'-, 4'-, and 6'-deoxy derivatives and the 3-O-methyl derivative of methyl beta-lactoside have been synthesised and their binding to the galactose-specific agglutinin from Ricinus communis (RCA-120) has been investigated. The results indicate that HO-3,4,6 of the beta-D-galactopyranose moiety are the key polar groups. The main difference from the closely related ricin lectin RCA-60 involves HO-6 of the D-glucopyranose moiety, which seems to contribute to the binding of the carbohydrate to RCA-60 but not to RCA-120.     

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.     

Expression of lectin-binding surface glycoproteins during the development of Schistosoma mansoni schistosomula

Tegumental glycoproteins of Schistosoma mansoni cercariae, mechanically produced 24-hr and 48-hr schistosomula, and adult worms were radioiodinated with the Bolton-Hunter reagent, then isolated by lectin affinity chromatography. SDS-PAGE revealed Con A binding glycoproteins with apparent molecular weights of 180,000, 150,000, 43,000, and 30,000 in detergent extracts of the tegument of cercariae. These glycoproteins are retained by 24-hr mechanically produced, cultured schistosomula and are accompanied by the appearance of 2 additional labeled glycoproteins, mol. wt. 66,000 and 57,000. In 48-hr schistosomula, there is a marked increase in the relative size of the 66,000 mol. wt. peak. In contrast, the 57,000 mol. wt. glycoprotein is the major radiolabeled Con A binding component of the adult tegument; the other peaks are either reduced or absent in adults. Similar findings were obtained following affinity chromatography using immobilized Lens culinaris lectin or Ricinus communis agglutinin, and following metabolic labeling of glycoproteins with tritiated galactose.     

Characterization of surface glycoproteins on Schistosoma mansoni adult worms by lectin affinity chromatography

Adult Schistosoma mansoni were radiolabeled by direct radioiodination using the Bolton-Hunter reagent or by metabolic labeling using radioactive hexose precursors. Tegumental material was extracted by freeze-thaw or by incubation in the non-ionic detergent Nonidet P-40, then applied to chromatography columns containing the following immobilized lectins: Con A, lentil lectin, wheat germ agglutinin, soybean agglutinin and the agglutinins from Ricinus communis and Helix pomatia. SDS-PAGE analysis of the sugar eluates from these columns revealed the presence of 15 glycoproteins with apparent molecular weights greater than or equal to 300,000, 215,000, 168,000, 152,000, 134,000, 122,000, 108,000, 83,000, 58,000, 53,000, 46,000, 41,000, 34,000, 30,000 and 23,500. Many of the glycoproteins reacted with more than one lectin. Information about carbohydrate content and lectin binding provides a preliminary characterization of the tegumental glycoprotein antigens of adult worms.     

Carbohydrate Composition of Variant-Specific Surface Antigen Glycoproteins from Trypanosoma brucei

SYNOPSIS. The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetyl-glucosamine). The glycoprotein from variant 048, strain 427 contained (±20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an integral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin bands with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120,000).     

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.     

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two-Dimensional Electrophoresis and Identification by Lectin Binding

The proteins of highly purified chromaffin-granule membranes were separated by one- or two-dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organelles. Two-dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin-binding bands are discussed: neither cytochrome b561 nor the F1-like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin-A binding proteins, as well as dopamine beta-hydroxylase, are present in the chromaffin granule lysate.     

Lectin localization in human nerve by biochemically defined lectin-binding glycoproteins, neoglycoprotein and lectin-specific antibody

Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physiologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant beta-galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the beta-galactoside-specific plant lectins from Ricinus communis and Erythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes.

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).     

Identification and analysis of altered alpha1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis

Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.     

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins.

We have reported the isolation of a clone (termed 15B) of Chinese hamster ovary (CHO) cells which are deficient in certain plant lectin-binding sites and have decreased amounts of sialic acid, galactose, and N-acetylglucosamine in its membranes (Gottlieb et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1078-1082). This study demonstrates that extracts of 15B cells, in contrast to the parent cell line, do not transfer N-acetylglucosamine residues from UDP-GlcNAc to certain glycopeptide and glycoprotein acceptors containing terminal nonreducing alpha-linked mannose residues. The decreased enzyme activity could not be accounted for by the presence of inhibitors, altered pH, or Mn2+ requirements of the glycosyltransferase or increased N-acetylglucosaminidase activity in the extracts. The finding that the 15B cell extracts have significant but reduced N-acetylglucosaminyltransferase activity toward a degraded orosomucoid acceptor suggests that these cells have a selective loss of one of several specific N-acetylglucosaminyltransferases which are present in the parent CHO cells. The sialyl- and galactosyltransferase activities of 15B and parent CHO cells are comparable. Parent CHO and 15B cells were grown in radioactive glucosamine to label the membrane glycoproteins. Solubilization of these glycoproteins and passage over a Ricinus communis agglutinin I (RCA I) Sepharose affinity column revealed that no labeled 15B glycoprotein material bound, whereas 50 percent of the CHO membrane glycoproteins bound and could be eluted with the haptene lactose, demonstrating that 15B cells are virtually devoid of membrane oligosacharides capable of binding to the RCA I lectin. The 15B membrane glycoproteins exhibited a marked shift toward glycoprotein species of lower molecular weight when examined by gel electrophoresis in sodium dodecyl sulfate. It is proposed that this shift in the mobility of the 15B membrane glycoproteins results from a decreased glycosylation of a number of membrane glycoproteins relative to their counterparts in CHO cells. The deficient N-acetylglucosaminyltransferase activity in 15B cells can account for the decreased glycosylation of the 15B cell membrane glycoproteins.