Complex-type Carbohydrate Research Articles

Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein

Estrogen synthetase (aromatase) is a cytochrome P-450 enzyme system which converts androgens to estrogens. Although this enzyme has been purified and the cDNA-derived amino acid sequence elucidated, very little is known regarding post-translational modifications of this physiologically crucial enzyme. We show here that the cytochrome P-450 component, P-450 ES, purified from human term placental microsomes, is a glycoprotein based on the following evidence: its molecular weight is decreased following treatment with endoglycosidase F, concanavalin A-biotin specifically binds to this protein immobilized on nitrocellulose, and its oligosaccharide composition is consistent with a single N-linked fucosylated complex type carbohydrate chain. In a reconstitution system, the aromatase activity using the endoglycosidase F-treated P-450 ES was reduced by about 35–40% relative to the native form, regardless of whether androstenedione or testosterone was used as substrate.

Intracellular processing of apolipoprotein J precursor to the mature heterodimer.

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.

Interactions of asparagine-linked carbohydrates with concanavalin A. Nuclear magnetic relaxation dispersion and circular dichroism studies

By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.

Recombinant carbohydrate variant of human choriogonadotropin beta-subunit (hCG beta) descarboxyl terminus (115-145). Expression and characterization of carboxyl-terminal deletion mutant of hCG beta in the baculovirus system.

A recombinant analog of human choriogonadotropin beta-subunit descarboxyl-terminal peptide (115-145 residues, delhCG beta) was obtained by the expression of corresponding beta cDNA in the baculovirus expression system. The efficiency of expression and secretion was high. The recombinant delhCG beta was purified by immunoaffinity using a specific monoclonal antibody against hCG beta and reverse phase high performance liquid chromatography. The hCG beta analog lacked the carboxyl-terminal 31-residue peptide as well as the four O-linked carbohydrates. Also, the N-linked "complex" type carbohydrates in the deletion mutant were modified to the high mannose type. The apparent molecular weights of delhCG beta in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions were found to be 19,000 and 27,500 respectively. delhCG beta on hydrolysis with endo N-acetylglucosaminidase F or H yielded a 17,500 protein band whereas treatment with N-glycanase gave a protein band with a molecular weight of 16,000. The carbohydrate analysis of delhCG beta, calculated on the basis of 4 residues of N-acetylglucosamine, showed 3 or 4 fucose, 0.6 N-acetylgalactosamine, and 11.4 mannose residues, indicating the high mannose type structures of the two N-linked carbohydrate chains. Despite the carbohydrate modification of the N-linked carbohydrates and the carboxyl-terminal deletion, the delhCG beta had about 87% of the immunological activity of the native hCG beta, indicating no significant conformational alteration induced by the mutation. The delhCG beta combined readily with native hCG alpha, and the reconstituted hCG alpha del beta required 0.031 pmol to achieve 50% inhibition of binding of the tracer with rat lutropin/choriogonadotropin receptor compared with 0.039 pmol by native hCG. Like native hCG, hCG alpha del beta also had most comparable ability to stimulate cAMP accumulation and progesterone production in rat Leydig cells. Thus it is clear from the data that the carboxyl-terminal deletion and thereby the deletion of four O-linked carbohydrates had no effect on its in vitro immunological and biological properties.

Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line

The oligosaccharide structures linked to Asn289 of a recombinant (r) variant (R561S) human plasminogen (HPg) expressed in Chinese hamster ovary (CHO) cells, after transfection of these cells with a plasmid containing the cDNA coding for the variant HPg, have been determined. Employing high-performance anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the protein by glycopeptidase F, compared with elution positions of standard oligosaccharides, coupled with monosaccharide compositional determinations and analyses of sequential exoglycosidase digestions and specific lectin binding, we find that considerable microheterogeneity in oligosaccharide structure exists at this sole potential N-linked glycosylation site on HPg. A variety of high-mannose structures, as well as bi-, tri-, and tetraantennary complex-type carbohydrate, has been found, in relative amounts of 1-25% of the total oligosaccharides. The complex-type structures contain variable amounts of sialic acid (Sia), ranging from 0 to 5 mol/mol of oligosaccharide in the different glycan structures. Neither hybrid-type molecules, N-acetylglucosamine bisecting oligosaccharides, nor N-acetyllactosaminyl-repeat structures were found to be present in the complex-type carbohydrate pool in observable amounts. Of interest, a significant portion of the Sia exists an outer arm structures in an (alpha 2,6) linkage to the penultimate galactose, a novel finding in CHO cell-directed glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase fromFlavobacterium meningosepticum

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F) from Flavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N (asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with human alpha 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of human alpha 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.

Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.

Microheterogeneity of the carbohydrate moiety of the human erythrocyte glucose transporter

The carbohydrate moiety of the human erythrocyte glucose transporter was isolated using two independent methods: hydrazinolysis andN-glycanase treatment. The major structure observed was constituted of complex-type carbohydrate chains carrying repetitive units ofN-acetyllactosamine. This structure exhibited microheterogeneity: a broad variability in the number of repetitive units, presence of branched structures and substitution by fucosyl residues. Moreover, significant amounts of bi-antennary and hybrid structures were present.

Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes

We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH 2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2,respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possesses one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numberous structural similarities.

Characterization of a Thyroid Sulfotransferase Responsible for the 3-O-Sulfation of Terminal β-D-Galactosyl Residues in N-Linked Carbohydrate Units

Calf thyroid microsomes were found to contain an enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAPS) to C-3 of terminal galactose residues in beta 1----4 linkage to GlcNAc. This sulfotransferase is believed to be involved in the biosynthesis of the recently described Gal(3-SO4) capping groups present in the N-linked oligosaccharides of thyroglobulin (Spiro, R.G., and Bhoyroo, V. D. (1988) J. Biol. Chem. 263, 14351-14358). Assays with various native and modified glycopeptides indicated that the enzyme acted optimally on complex-type carbohydrate units in which beta-linked Gal has been uncovered by desulfation or brought into a terminal position by removal of sialyl and/or alpha-galactosyl residues. With fetuin asialoglycopeptides as acceptors (Km = 0.1 mM) the transfer of sulfate from PAPS (Km = 6.3 microM) had a pH optimum of approximately 7.0, required Mn2+ ions (10-50 mM) and was markedly stimulated by Triton X-100 (0.1%) and ATP (2 mM). The same enzyme apparently sulfated free N-acetyllactosamine (LacNAc; Km = 0.69 mM) and its ethyl glycoside, indicating that it had no absolute requirement for a peptide recognition site. Studies with a number of disaccharides related to LacNAc provided information relating to the specifying role of the beta 1----4 galactosyl linkage and the configuration at C-2 of the sugar to which it is attached. Hydrazine-nitrous acid-NaBH4 treatment of the 35S-labeled products from sulfotransferase action on asialoglycopeptides as well as on the ethyl glycoside of LacNAc yielded the same disaccharide, Gal(3-SO4) beta 1----4 anhydromannitol, as is obtained from a similar treatment of thyroglobulin. Subcellular distribution studies indicated that the PAPS:galactose 3-O-sulfotransferase is located in the Golgi compartment which is consistent with the late occurrence of the requisite beta-galactosylation step. It is proposed that in certain tissues the ultimate nature of the capping groups attached to glycoproteins containing terminal Gal beta 1----4GlcNAc sequences could be the result of a competition between this 3-O-sulfotransferase and sialyl- and/or alpha-galactosyltransferases.

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron, E. cristagalli, E. flabelliformis, andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certain Erythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 27:1034-41]. The present work describes a comparative study of the binding and precipitating activities of four Erythrina lectins, viz., E. corallodendron, E. cristagalli, E. flabelliformis, and E. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, the Erythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.

Novel glycosylation pathways of retroviral envelope proteins identified with avian reticuloendotheliosis virus.

Previously, we identified two mature glycoproteins, gp90, the surface glycoprotein, and gp20, the transmembrane protein, from avian reticuloendotheliosis virus and an avian reticuloendotheliosis virus env gene-encoded intracellular polyprotein gPr77env, but the precise relationship of gPr77env to the mature envelope proteins was not determined (W.-P. Tsai, T.D. Copeland, and S. Oroszlan, Virology 155:567-583, 1986). In the present study, using metabolic labeling of viral proteins with [35S]cysteine, radioimmunoprecipitation, and carbohydrate structure analysis, we have identified a higher-molecular-weight endo-H-resistant env gene-encoded polyprotein designated gPr115env in addition to the endo-H-sensitive gPr77env. It appears that gPr77env is the primary polyprotein precursor, modified with mannosyloligosaccharides that are processed into sialic-acid-rich extraordinarily large complex-type carbohydrates (up to 17 kilodaltons for each N-linked site) on the gp90 domain but not on the gPr22 domain. In this process, gPr77env is converted into the apparently endo-H-resistant secondary polyprotein, gPr115env, which is rapidly processed into gp90 and gPr22. The proteolytic processing which occurs only after the appearance of an endo-H resistant precursor is now clearly demonstrated for a retrovirus. Some important aspects of carbohydrate structure, including the site-specific glycosylation, as well as the intracellular location and nature of the potential enzyme involved in the proteolytic cleavage of gPr115env are discussed.

Insulin-like growth factor I receptors on mouse neuroblastoma cells. Two beta subunits are derived from differences in glycosylation.

We have characterized receptors for the insulin-like growth factor (IGF-I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell-free system, IGF-I and insulin stimulated the phosphorylation of 95-kDa and 105-kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF-I receptor beta subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor beta subunit is a separate single 95-kDa protein. Enzymatic digestion of IGF-I receptor beta subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS-PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N-linked complex-type carbohydrate chains on the extracellular domain of beta subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion. Analysis of IGF-I receptor beta subunit subtypes in these cells may provide insights into the mechanism of action of IGF-I on neural tissues.

Identification, purification, and characterization of a stilbenedisulfonate binding glycoprotein from canine kidney brush border membranes. A candidate for a renal anion exchanger.

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose, bisected hybrid, and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.

Identification and Characterization of an Estrogen-Inducible Glycoprotein (Uterine Secretory Protein-1) Synthesized and Secreted by Rat Uterine Epithelial Cells*

An estrogen-inducible 97-kilodalton (kDa) secretory glycoprotein, designated as uterine secretory protein (USP)-1, synthesized by rat uterine epithelial cells was identified and characterized. Uterine luminal fluid (ULF) proteins were labeled by direct administration of [35S]methionine into uterine lumen of rats. Incorporation of [35S]methionine into ULF proteins was negligible in ovariectomized rats. However, when 17 beta-estradiol (E2) was administered in the ovariectomized rat as sc paraffin pellets, a marked increase of [35S]methionine incorporation was noted after 2 days of treatment, showing that the de novo synthesis and secretion of ULF proteins were induced by E2. Six estrogen-inducible polypeptides (130, 110, 97, 65, 42, and 39 kDa) were identified in the analysis of the labeled ULF proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Four of these polypeptides (110, 65, 42, and 39 kDa) were adsorbed by the immobilized antibody against rat serum proteins, indicating that these polypeptides are antigenically similar to serum proteins. Two polypeptides (130 and 97 kDa) not adsorbed by the antibody column were suggested to be uterine-specific secretory proteins. The 97-kDa protein (USP-1) was further purified by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis and electroelution. Reactivity of purified USP-1 to various lectins and carbohydrate composition analysis suggested that USP-1 possesses biantennary N-linked complex-type carbohydrate chain with fucose. Rabbit polyclonal antibody which can specifically immunoprecipitate [35S]methionine-labeled USP-1 was developed. Dot blot enzyme immunoassay showed that 4-day E2-treated ULF contains 78.3 +/- 24.1 (+/- SE) micrograms USP-1/mg protein. Immunohistochemical staining of rat uterine tissue showed that this protein localized only in the epithelial cells treated with estrogen (E2 and diethylstilbestrol). Testosterone, progesterone, and dexamethasone failed to induce synthesis and secretion of USP-1 as assessed by dot blot enzyme immunoassay, immunoprecipitation of [35S]methionine-labeled ULF proteins, and immunohistochemical staining of uterine tissue. The present result, for the first time, revealed that estrogen can induce synthesis and secretion of specific secretory protein which could be the useful marker to analyze the molecular mechanism of estrogen action in rat uterine epithelial cells.

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors.

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides.

In the preceding paper (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293), we have demonstrated that certain high mannose and bisected hybrid type glycopeptides are bivalent for concanavalin A (ConA) binding. In the present study, we have investigated the interactions of ConA with a series of synthetic nonbisected and bisected complex type oligosaccharides and related glycopeptides. The modes of binding of the carbohydrates were studied by nuclear magnetic relaxation dispersion techniques, and their affinities were determined by hemagglutination inhibition measurements. We find that certain bisected complex type oligosaccharides are capable of binding and precipitating the lectin. The corresponding nonbisected analogs, however, bind but do not precipitate the protein. The stoichiometries of the precipitin reactions were investigated by quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that the bisected complex type oligosaccharides are bivalent for lectin binding. Data for the nonbisected analogs are consistent with their being univalent. The nuclear magnetic relaxation dispersion and precipitation data indicate that nonbisected and bisected complex type carbohydrates bind with different mechanisms and conformations. The former class binds by extended site interactions with the protein involving the 2 alpha-mannose residues on the alpha(1-6) and alpha(1-3) arms of the core beta-mannose residue. The latter class binds by only 1 of these 2 mannose residues, which leaves the other mannose residue free to bind to a second ConA molecule. The role of the bisecting GlcNAc residue in affecting the binding properties of complex type carbohydrates to ConA is discussed, and the results are related to the possible structure-function properties of complex type glycopeptides on the surface of cells.

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.

Glycosylation of the bovine coronavirus hemagglutinin protein.

The bovine coronavirus hemagglutinin protein gp140 is composed of disulfide-linked subunits of 65 kDa. This protein was further characterized with regard to its glycosylation. The glycosylated subunits of the protein are polypeptides having a molecular mass of 42.5 kDa. Both subunits appear to be processed to the same extent by the addition of N-linked oligosaccharides. Each subunit on the mature virion has 6-7 high mannose and 3-4 complex type carbohydrate chains attached to it.