N-linked Carbohydrate Chains Research Articles

The mammalian beta-adrenergic receptor: structural and functional characterization of the carbohydrate moiety.

Mammalian beta-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr approximately 64,000. Treatment of purified [125I]-labeled hamster lung beta-adrenergic receptor with alpha-mannosidase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endoglycosidase F results initially in the formation of a Mr approximately 57,000 peptide which is further converted to Mr approximately 49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (approximately 10% of which are fucosylated) on approximately 45% of the receptors. The remaining approximately 55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of beta-adrenergic agonists and antagonists. Moreover, the ability of control, alpha-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, alpha-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleotide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the beta-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.

Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate.

Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets.

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.

Enzymatic deglycosylation of thyroid-stimulating hormone with peptide N-glycosidase F and endo-β-N-acetylglucosaminidase F

We investigated the ability of two enzymes, peptide N-glycosidase F (PNGase F) and endo-β-N-acetylglucosaminidase F (Endo F), to deglycosylate microgram quantities of bovine TSH and its subunits under nondenaturing conditions. One oligosaccharide chain could be selectively removed from the α subunit by PNGase F, and all the oligosaccharide chains from both subunits could be removed by Endo F. These methods of enzymatic deglycosylation should permit study of the functional role of each N-linked carbohydrate chain of various glycoprotein hormones.

Identification of a α-NeuAc-(2→3)-β- d-galactopyranosyl N-acetyl-β- d-galactosaminyltransferase in human kidney

Microsomal preparations from human kidney were found to contain enzymic activity capable to transfer N-acetylgalactosamine from UDP- N-acetylgalactosamine to native bovine fetuin. The acceptor structures on the fetuin molecules were identified as N- as well as O-linked glycans with a markedly higher incorporation into the N-linked carbohydrate chains. Analysis of the alkali-labile transferase products by thin-layer chromatography indicated that the enzyme is able to synthesize structures having mobilities identical with those found on glycophorin from Cad erythrocytes. Mild acid treatment and enzymic hydrolysis with N-acetylhexosaminidase from jack beans of the N-linked transferase products suggested that β- d-Gal pNAc-(1→4)-[α-NeuAc-(2→3)]-β- d-Gal p-(1-ar structures were formed by the enzymic reaction on both N- and O-linked acceptors. The enzyme might, therefore, be involved in the biosynthesis of Sd a (and Cad) antigenic structures. By use of various oligosaccharides, glycopeptides, and glycolipids having well characterized carbohydrate sequences, the acceptor-substrate specificity of the N-acetylgalactosaminyltransferase was determined. The enzyme generally recognized α-NeuAc-(2→3)-β- d-Gal groups as acceptors, but in a certain conformation. Thus, tri- and tetra-saccharide alditols, native human glycophorin A, and GM 3 were not acceptor substrates although they carry the potential disaccharide acceptor unit. When these structures were presented as sialyl-(2→3)-lactose or as a tryptic peptide from glycophorin A, they were shown to be rather good acceptor substrates for the N-acetyl-β- d-galactosaminyltransferase from human kidney.

Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid-linked carbohydrate chains.

Biochemical, immunological, and genetic techniques were used to investigate the genetic defects in three types of low density lipoprotein (LDL) receptor-deficient hamster cells. The previously isolated ldlB, ldlC, and ldlD mutants all synthesized essentially normal amounts of a 125,000-D precursor form of the LDL receptor, but were unable to process this receptor to the mature form of 155,000 D. Instead, these mutants produced abnormally small, heterogeneous receptors that reached the cell surface but were rapidly degraded thereafter. The abnormal sizes of the LDL receptors in these cells were due to defective processing of the LDL receptor's N- and O-linked carbohydrate chains. Processing defects in these cells appeared to be general since the ldlB, ldlC, and ldlD mutants also showed defective glycosylation of a viral glycoprotein, alterations in glycolipid synthesis, and changes in resistance to several toxic lectins. Preliminary structural studies suggested that these cells had defects in multiple stages of the Golgi-associated processing reactions responsible for synthesis of glycolipids and in the N-linked and O-linked carbohydrate chains of glycoproteins. Comparisons between the ldl mutants and a large number of previously isolated CHO glycosylation defective mutants showed that the genetic defects in ldlB, ldlC, and ldlD cells were unique and that only very specific types of carbohydrate alteration could dramatically affect LDL receptor function.

Oligomannoside-type glycopeptides inhibiting adhesion of Escherichia coli strains mediated by type 1 pili: preparation of potent inhibitors from plant glycoproteins.

Various structurally defined glycopeptides of natural origin were tested as inhibitors of guinea pig erythrocyte agglutination by enteropathogenic Escherichia coli strains expressing type 1 pili. Besides hybrid-type glycoasparagines from ovalbumin which were not active, large oligomannoside-type carbohydrate chains from legume storage glycoproteins moderately inhibited hemagglutinations, whereas the short oligomannoside-type glycoasparagine from ovalbumin Man alpha(1----6) [Man alpha(1----3)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc beta(1----4)GlcNAc beta(1----N)Asn exhibited a potent activity. These results strongly suggested that the nonsubstitution of the alpha(1----3)-linked mannosyl residue from the N-linked glycopeptide core structure is the key determinant in the minimal structural requirement specific to this fimbrial lectin. Such Man5GlcNAc2-containing glycopeptides were obtained from larger N-linked carbohydrate chains, occurring abundantly in natural sources. The ability of jack bean alpha-mannosidase to cleave the alpha(1----2)-linked mannoses more rapidly than the others allowed the controlled digestion of large oligomannoside-type glycopeptides from legume storage glycoproteins. Such shortened glycopeptides of plant origin were prepared which strongly inhibited guinea pig erythrocyte agglutinations as well as bacterial adhesion on human buccal cells, thus confirming their similarity (if not identity) with the receptor of type 1 pili on mammalian cells. The importance of this preparation of a receptorlike compound that inhibits bacterial adhesion with regard to the research on the role of type 1 pili in E. coli pathogenicity is discussed.

Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein.

The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of attachment sites for N-linked carbohydrate groups of class II histocompatibility α-chain and analysis of possible O -linked glycosylation of α- and γ -chains

Two glycopeptide fractions were isolated from a tryptic digest of the human class II antigen α-subunit, by chromatography on Lens culinaris and Ricinus communis lectin columns, respectively. Partial NH 2-terminal sequence analysis of radiochemically labelled glycopeptide fractions allowed alignment with two stretches of the deduced DRα sequence, each encompassing a signal for N-linked glycosylation, i.e. Asn-X-Thr(Ser). The fraction displaying affinity for L. culinaris lectin was susceptible to the action of endo-β- N-acetylglucosaminidase H whereas the fraction adsorbed to R. communis was not. Since DRα is known to carry only one N-linked carbohydrate chain in the high-mannose form, this glycan could thereby be mapped to Asn 78. Accordingly, the complex N-linked carbohydrate is attached to Asn 118. Moreover, analysis of material released from class II antigens and γ-chains upon mild alkaline hydrolysis, indicates the presence of O-linked sugars on the α- and γ- but not the β-subunits.

Sequence analysis of the membrane protein V3 of the flavivirus west nile virus and of its gene

Flaviviruses contain a large membrane-associated protein V3, having a mol mass of about 50 kDa which is responsible for hemagglutination. We have isolated the V3 protein from the West Nile (WN) flavivirus and determined its amino-terminal amino acid sequence and amino acid sequences of fragments derived from this protein. We have also transcribed parts of the WN virus genome RNA into cDNA and cloned and sequenced this CDNA. The results of these analyses have allowed us to identify the region of the viral genome coding for the V3 protein. In this report we describe the total nucleotide sequence of the genome region coding for the WN virus V3 protein and the amino acid sequence of the V3 protein derived from these analyses. The exact carboxy terminus of the V3 protein has not been determined in these experiments. These analyses have shown that the V3 protein of WN virus does not contain an Asn-X-Ser/Thr sequence which could allow addition of N-linked carbohydrate chains to this protein. In accordance with this finding, analyses of metabolic labeling of the V3 protein using [ 3H]glucosamine indicate that the WN virus V3 protein is an unglycosylated protein. Together with our earlier analyses these results show that the viral structural proteins are present on the genome RNA in the order 5′-terminuscore protein (V2)-small membrane-associated protein (NV2)-large membrane-associated protein (V3) and describe the nucleotide sequences coding for all WN virus structural proteins identified so far. A hypothesis concerning the processes involved in the synthesis of all viral structural proteins and the probable orientation of these proteins relative to the endoplasmatic reticulum membrane based on the structure of these proteins is discussed.

The protein component of scrapie-associated fibrils is a glycosylated low molecular weight protein.

Scrapie-associated fibril protein (SAF-protein) extracted from infectious scrapie-associated fibrils (SAF) isolated from scrapie hamster brains is not infectious. SAF-protein is composed of various mol. wt. species of glycoproteins differing in carbohydrate content rather than amino acid composition. The N-linked carbohydrate chains represent approximately 40-60% of the mol. wt. of SAF-protein. The deglycosylated SAF-protein has a surprisingly low mol. wt. of approximately 7 kd, representing approximately 55 amino acid residues. This size and chemical analyses indicate that SAF-protein is an amyloid-type of protein. The simplest explanation for the available data is that SAF-polypeptide is very likely not to be part of the scrapie agent but that it is, like other amyloid proteins, derived from host-encoded proteins and not infectious. It is suggested that the infectivity of fractions rich in SAF is due to co-purification of scrapie virus and SAF caused by the high carbohydrate content of SAF-protein.

Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-β- N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, α-fucose, α-galactose, and α- N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.

Binding Characteristics of 3H-Dihydroalprenolol to β-Adrenergic Receptors of Rat Brain: Influence of Lectins

The significance of the carbohydrate moieties of the β-adrenergic receptor molecule in the rat brain was examined using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of lectins, ccncanavlin A (Con A), Phaseolus vulgaris agglutinin (PHA), and wheat germ agglutinin (WGA) on the affinity of the β-adrenoceptor. The rat brain was used and the β-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a iigand. Con A and PHA significantly caused an increase in the values of the density of β-adrenoceptor (Bmax) and a reduction in the values of the dissociation constant (Kd), but significant changes were not observed with WGA. These results strongly suggest that the carbohydrate moieties of the cell surface containing the β-adrenoceptor molecule may have a crucial role in the drug-receptor interaction, and they imply that the β-adrenoceptor molecule is a glycoprotein which contains N-linked carbohydrate chains.

Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of lectins.

The significance of the carbohydrate moieties of the beta-adrenergic receptor molecule in the rat brain was examined using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of lectins, concanavalin A (Con A), Phaseolus vulgaris agglutinin (PHA), and wheat germ agglutinin (WGA) on the affinity of the beta-adrenoceptor. The rat brain was used and the beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a ligand. Con A and PHA significantly caused an increase in the values of the density of beta-adrenoceptor (Bmax) and a reduction in the values of the dissociation constant (Kd), but significant changes were not observed with WGA. These results strongly suggest that the carbohydrate moieties of the cell surface containing the beta-adrenoceptor molecule may have a crucial role in the drug-receptor interaction, and they imply that the beta-adrenoceptor molecule is a glycoprotein which contains N-linked carbohydrate chains.

Binding Characteristics of 3H-Dihydroalprenolol to β-Adrenergic Receptors of Rat Brain: Influence of Exo- and Endo-Glycosidases and Glycopeptidase

The significance of carbohydrate moieties containing the β-adrenoceptor molecule in the rat brain was examined using radioligand binding assay methods. Thus, this experiment was designed to assess the effects of exoglycosidase (α-D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and endoglycosidase H), and glycopeptidase A on the affinity of β-adrenoceptor. The main reason why five kinds of enzymes were used in the present study is that they can hydrolyze different carbohydrate molecules from cell membranes. Rat brain was used and β-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to β-adrenoceptors was sensitive to very low concentration of endoglycosidase H and glycopeptidase A, thus indicating that the treatments with these enzymes of rat brain membrane appear to decrease the number of β-receptor binding sites. On the other hand, the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the membrane induced lower values of the dissociation constant (Kd) than those of the control, α-D-mannosidase and endoglycosidase D are without effect in spite of the removal of hexose contents and total carbohydrate contents with these treatments, respectively. These results imply that complex type N-linked acidic carbohydrate chains containing neuraminic acid and high mannose type N-linked carbohydrate chains, which are hydrolyzed with endoglycosidase H and glycopeptidase A, of the rat brain membrane containing β-adrenoceptor molecules play a crucial role in the drug-receptor interaction.

Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of exo- and endo-glycosidases and glycopeptidase.

The significance of carbohydrate moieties containing the beta-adrenoceptor molecule in the rat brain was examined using radioligand binding assay methods. Thus, this experiment was designed to assess the effects of exoglycosidase (alpha-D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and endoglycosidase H), and glycopeptidase A on the affinity of beta-adrenoceptor. The main reason why five kinds of enzymes were used in the present study is that they can hydrolyze different carbohydrate molecules from cell membranes. Rat brain was used and beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to beta-adrenoceptors was sensitive to very low concentration of endoglycosidase H and glycopeptidase A, thus indicating that the treatments with these enzymes of rat brain membrane appear to decrease the number of beta-receptor binding sites. On the other hand, the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the membrane induced lower values of the dissociation constant (Kd) than those of the control. alpha-D-mannosidase and endoglycosidase D are without effect in spite of the removal of hexose contents and total carbohydrate contents with these treatments, respectively. These results imply that complex type N-linked acidic carbohydrate chains containing neuraminic acid and high mannose type N-linked carbohydrate chains, which are hydrolyzed with endoglycosidase H and glycopeptidase A, of the rat brain membrane containing beta-adrenoceptor molecules play a crucial role in the drug-receptor interaction.

Changes in cell-surface sialic acid content during chick embryo development

The amount of cell-surface sialic acid found in the material released by trypsin from chick fibroblasts rose markedly from day 8 to day 16 of embryo developmet. The rise seemed to result from two processes: increased sialylation of N-linked carbohydrate chains, and enhancement of the amount of O-linked structures. Eight-day cells were more quickly detached from the substrate than 16-day cells, since in 8-day fibroblasts detachment of half the adherent cells only required 10 min, compared to 20 min in 16-day fibroblasts. Re-adhesion to the substrate was also faster for the younger fibroblasts, and 40% of the 8-day trypsin-treated cells re-adhered within 30 min compared to about 3 h for the same proportion of 16-day cells. The age-dependent differences in the amount of cell-surface sialic acid do not account for the differences in the adhesive capacity of embryo cells. The fact that neuraminidase did not affect cell detachment or re-adhesion indicates that cell-surface sialic acid does not play an important part in the adhesive capacity of embryo fibroblasts.

Biosynthesis of the major glycoprotein associated with zymogen-granule membranes in the pancreas.

The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr = 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-beta-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides. Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5-6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73000-61000. We conclude that GP-2 contains five or six N-linked carbohydrate chains. From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (greater than 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (less than 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.

Biochemical characterization of an invariant polypeptide associated with Ia antigens in human and mouse

I i a 31,000 mol. wt polypeptide chain associated with murine and human Ia antigens was investigated for its labeling pattern, carbohydrate content and structural polymorphism. Two-dimensional gel electrophoretic analysis of tunicamycin treated cells from mouse and human lymphocytes shows that I i contains two N-linked carbohydrate chains. I i is a methionine rich polypeptide. Tryptic and chymotryptic two dimensional peptide maps of I i chain associated with I- A and I- E subregion products are identical. This absence of polymorphism holds true when I i chain is isolated from different mouse haplotypes. Human I i chains from different HLA-DR types appear also invariant by peptide map analysis. By molecular weight, carbohydrate content, charge and tryptic and chymotryptic maps criteria, I i of mouse and human are strikingly homologous.

Carbohydrate determinants involved in both the binding and action of insulin in rat adipocytes

The insulin receptor apparent affinity was markedly decreased in fat cells treated with lectins specific either for d-galactose ( Ricinus communis agglutinin I, RCA I), d-mannose (concanavalin A, Con A, Lens culinaris agglutinin, LCA) or N-acetyl- d-glucosamine (wheat germ agglutinin, WGA), as indicated by a rightward shift of the binding competition curves and almost lineared Scatchard plots. Limulus polyphemus agglutinin (LPA), specific for sialic acid, was ineffective. All lectins enhanced 2-deoxy- d-glucose uptake with relative bioactivities (maximal lectin effect/maximal insulin effect) of 68–86%. Insulin and lectin stimulatory effects were antagonized by specific carbohydrates used as competitors and inhibited by cytochalasin B (70 μM). Maximal effects of insulin and lectins were not additive and were completely abolished in neuraminidase-treated fat cells. Lectins did not affect insulin degradation. These data show that sialylated glycosidic moieties containing d-galactose, d-mannose and N-acetyl- d-glucosamine units are involved in both processes of insulin ‘high affinity’ binding and activation of glucose transport but are not implicated in hormone degradation. They suggest that N-linked carbohydrate chains of the complex type may be essential for functional insulin receptor and post-receptor systems.