Structural studies on the carbohydrate moieties of human liver alpha-L-fucosidase

alpha-L-Fucosidase was purified from human liver to apparent homogeneity and subjected to exhaustive digestion with Pronase. The resulting glycopeptides were isolated by gel filtration on Sephadex G-50 and further fractionated by Bio-Gel P-4 chromatography. Five glycopeptide fractions were obtained. The structures of the carbohydrate portions of all glycopeptide components were fully characterized by a combination of 500-MHz 1H NMR spectroscopy and carbohydrate composition analysis. Fraction I contained disialyl diantennary glycopeptides of the N-acetyllactosamine type. Fractions II and III contained predominantly mono(sialyl-N-acetyllactosaminyl) diantennary glycopeptides with the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----2) branch attached to alpha(1----3)-linked Man in II and to alpha(1----6)-linked Man in III. The N-acetyllactosamine-type glycopeptides in fractions I to III have a small portion (10-15%) of their Asn-linked GlcNAc residues substituted by additional alpha(1----6)-linked Fuc. Also, a minor portion of the NeuAc residues appeared to be attached to Gal in alpha(2----3) rather than alpha(2----6) linkage. Fraction IV contained a mixture of larger-size oligomannoside-type glycopeptides with a variable number (6 to 9) of Man residues. Smaller-size oligomannoside-type glycopeptides were found in fraction V, containing 3 or 5 Man residues; a small portion (10%) of the Man3GlcNAc2Asn component appeared to contain in addition a Fuc residue in alpha(1----6) linkage to the Asn-bound GlcNAc. The overall ratio of oligomannoside-type to N-acetyllactosamine-type carbohydrate structures was found to be 5:4. This article is the first account of the complete characterization of the oligomannoside-type structures in alpha-L-fucosidase; furthermore, the occurrence in alpha-L-fucosidase of mono(sialyl-N-acetyllactosaminyl) structures, Fuc-containing oligosaccharides, and NeuAc alpha(2----3) linked to Gal are reported for the first time.     

The structures of the carbohydrate moieties of the alpha subunit of human chorionic gonadotrophin.

The alpha subunit of human chorionic gonadotrophin was reduced with dithiothreitol followed by carboxymethylation with iodoacetic acid. The modified glycoprotein was hydrolysed with trypsin to give various peptides, the identities of which were established, and glycopeptides. The glycopeptides were separated by gel filtration and ion-exchange chromatography; they were subjected to component analysis and were found to represent the two carbohydrate moieties in the parent glycoprotein. Sequential removal with glycoside hydrolases of monosaccharide units from the glycopeptides demonstrated (1) that galactose, mannose, glucosamine (2-amino-2-deoxyglucose) and neuraminic acid (5-amino-3,5-dideoxy-glycero-galacto-2-nonulosonic acid) residues possess the D configurations, (2) that the glucosamine units are N-acetylated and (3) the order of the monosaccharide units in the chain, the neuraminic acid units being furthest from the peptide backbone of the subunit and substituting the D-galactose units. Methylation analysis of the glycopeptides by adaptation of the Hakomori technique demonstrated that: (4) D-galactose, D-mannose and N-acetylglucosamine (2-acetamido-2-deoxy-D-glucose) units exist in the pyranose forms; (5) the D-galactopyranose units are linked in the 1 and 6 positions; (6) the D-mannopyranose units exist in several forms, one in a terminal non-reducing position, one as 1,2-linked residues and some as 1,6-linked branch points; (7) the N-acetylglucosamine units are 1,6-linked. On the basis of the results of methylation and enzymic analysis, structures are proposed for the carbohydrate moieties and the assignments are compared with other data previously obtained by periodate-oxidation studies [Kennedy et al. (1974) Carbohydr. Res. 36, 369-377].     

Comparative study of the carbohydrate moieties of normal and pathological human immunoglobulins M.

The well-known heterogeneity of normal and pathological immunoglobulins M was investigated in a study involving the characterization of their carbohydrate moieties. Oligosaccharide units were released from the native molecule by hydrazinolysis, and they were fractionated by affinity chromatography on a concanavalin A-Sepharose column to yield separate N-acetyl-lactosaminic-type and oligomannosidic-type structures. Further identification of these oligosaccharides was attempted by t.l.c. on silica gel and by determination of their monosaccharide compositions. A comparative study of the oligosaccharide units belonging to each population of immunoglobulin M was possible. Similarities were found in the occurrence of both types of oligosaccharide structures, and, in addition, a common double heterogeneity could be demonstrated for N-acetyl-lactosaminic-type structures: they could be resolved by affinity chromatography into bi-, tri- and tetra-antennary structures, and they also showed differences in N-acetylneuraminic acid content. Though some variations were observed in the exact composition of the oligosaccharide units within each population, it was possible to consider a representative oligosaccharide-unit composition of normal immunoglobulin M as a standard for comparison. On this basis a predominance of multi-antennary structures was observed in the more glycosylated pathological immunoglobulins M (10% carbohydrate content), whereas oligomannosidic structures were increased in pathological immunoglobulins M with a lower content of carbohydrates (7%). These variations are thought to reflect differences in the biosynthetic processing pathway of the carbohydrate units of the pathological immunoglobulins M or the enhanced expression of a molecular clone.     

Investigations on the carbohydrate moieties of glycoprotein allergens

Many allergens are glycoproteins and their carbohydrate structure can contribute to the IgE reactivity. Therefore it is of great interest to study the carbohydrate structures of these particular antigens. Here, we present an overview of methods combining basic procedures in glycochemistry with various applications of electrophoresis that allow investigating single allergens in crude extracts. Various allergen extracts, e.g. from tomato, grass pollen and bacteria were analysed and the suitability of the tests are discussed.     

The structures of the carbohydrate moieties of bovine blood coagulation factor X

Bovine blood coagulation factor X contains both asparagine-linked and threonine-linked oligosaccharides. The asparagine-linked chain is a mixture of a tridecasaccharide NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and a dodecasaccharide NeuAc alpha 2 leads to 6 Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and their partial desialylation products. The threonine-linked chain is a mixture of NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, NeuGly alpha 2 leads to 3Gal beta 1 leads to 3 (NeuAc alpha 2 leads to 6)GalNAc, and NeuGly alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, and their partial desialized forms. The carbohydrate moieties of the factor X subgroups, factors X1 and X2, are identical.     

Differential accessibility of the carbohydrate moieties of Cls-Clr-Clr-Cls, the catalytic subunit of human Cl

The catalytic subunit of human Cl, Cls-Clr-Clr-Cls, is a Ca2+-dependent tetrameric association of two serine proteases, Clr and Cls, which are glycoproteins containing asparagine-linked carbohydrates. With a view to investigate the accessibility and the possible functional role of these carbohydrates, the isolated proteases and their Ca2+-dependent complexes were submitted to deglycosylation by peptide:N-glycosidase F, an endoglycosidase that specifically hydrolyzes all classes of N-linked glycans. Treatment of isolated Clr and Cls led to the removal of the carbohydrate moieties attached to their N-terminal alpha region, whereas those located in the C-terminal gamma-B catalytic domains were resistant to hydrolysis. Formation of the Ca2+-dependent Cls-Cls dimer and Cls-Clr-Clr-Cls tetramer induced specific protection of the single carbohydrate attached to the alpha region of Cls and of one of the two carbohydrates located in the corresponding region of Clr. Sequence studies indicated that the carbohydrates protected upon homologous (Cls-Cls) or heterologous (Clr-Cls) interactions are attached to asparagine residues 159 of Cls and 204 of Clr, at the C-terminal end of the EGF-like domain of both proteases. These data bring further evidence that Ca2+-dependent interactions between Clr and Cls are mediated by their N-terminal alpha regions and strongly suggest that, inside these regions, the EGF-like domains play an essential role in these interactions.     

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.     

A biological role of the carbohydrate moieties of laminin

The ways in which the carbohydrate moieties of laminin affect its cellular interactions have been examined by two different experimental approaches. In one approach, we used lectins in order to block specific carbohydrates on laminin which previously had been dried onto a plastic surface. We found that wheat germ agglutinin and Griffonia simplicifolia agglutinin I blocked the binding of the neuron-like rat pheochromocytoma cell line PC12. However, when concanavalin A was used cell binding was unaffected but neurite outgrowth was prevented, compared to controls, over a 24-h period. In the second approach we used unglycosylated laminin as a substratum on the plastic surface. We have developed a method for the purification of unglycosylated laminin from tunicamycin treated cultures of a mouse embryonal carcinoma derived cell line, M1536 B3, and have partially characterized the purified material. A mixture of unglycosylated and glycosylated laminin was selectively purified from the M1536 B3 cell lysate by an anti-EHS laminin monoclonal antibody immunoaffinity column. The unglycosylated laminin was separated from glycosylated laminin using G. simplicifolia lectin affinity chromatography. The lectins, wheat germ agglutinin, G. simplicifolia agglutinin I, and concanavalin A, did not bind to any of the subunits of unglycosylated laminin in Western blots. The unglycosylated laminin migrated as a single band in agarose-gel electrophoresis under nonreducing conditions indicating that it is a fully assembled and disulfide bonded molecule. Circular dichroism studies showed no differences between glycosylated and unglycosylated laminin, indicating similar molecular conformations. Western blots using antibodies specific for the A, B1, and B2 chains of laminin showed that unglycosylated laminin contained each of these subunits. We then performed cell binding and spreading or neurite outgrowth assays using unglycosylated laminin. A mouse melanoma cell line, B16 F1, bound to this laminin in the same numbers as to the control glycosylated laminin, but cell spreading was minimal. When this unglycosylated laminin was used as a substrate for PC12 cells neurite outgrowth was impaired; no effect was noted on the number of cells bound, compared to glycosylated laminin. We conclude from these results that once cells become bound to laminin the carbohydrate residues of that glycoprotein must be available to enable the cells to spread or to extend neurite processes.     

Structure of the carbohydrate moieties of bovine rhodopsin.

The sugar chains of bovine rhodopsin were released from the polypeptide moiety by hydrazinolysis and reduced with NaB[3H]4 after N-acetylation. The radioactive oligosaccharides thus obtained were fractionated into three components by paper chromatography. The structures of these components were elucidated as GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 and 6 Man alpha 1 leads to 6)Man beta leads to 4GlcNAc beta 1 leads to 4GlcNAc, and GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, by sequential exoglycosidase digestion, methylation analysis, and endo-beta-N-acetylglucosaminidase D digestion. The unusual features of the sugar chains of rhodopsin molecule seem to support the proposed processing pathway for the biosynthesis of asparagine-linked sugar chains of glycoproteins.     

Properties of human neutral bronchial mucins after modification of the peptide or the carbohydrate moieties.

Trypsin and pronase treatment of purified human neutral bronchial mucins released small fragments from the C-terminal end of these molecules and resulted in slight increases in their sedimentation coefficient presumably reflecting conformational changes. The antigenic determinant of neutral bronchial mucins which appears to be located on this C-terminal fragment is destroyed by pronase or by treatments such as periodate oxidation or galactose oxidase-bromine oxidation which modify the carbohydrate moieties. Thus, both amino acid and carbohydrate residues are involved in the structure of the antigenic determinant.     

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins

The pathways for protein N- and O-glycosylation in yeast cells are summarized. Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated. A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues. The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously. However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction. The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle. In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth. This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase. It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.     

Structural features of carbohydrate moieties in snake venom glycoproteins.

The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha-galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds.     

Studies on the unique presence of an N-acetylgalactosamine residue in the carbohydrate moieties of human follicle-stimulating hormone

The effect of cystine starvation on the transport system of cystine and glutamate was examined in cultures of human diploid fibroblasts. The 2-min uptake of cystine and glutamate increased progressively after a lag of 6 h of cystine starvation. There was approx. 2-3-fold increase, and the increased rate of uptake was accompanied by an increase in the Vmax and unchanged Km. The cystine starvation-induced enhancement appeared specific for the uptake of cystine and glutamate. Actinomycin D or cycloheximide completely blocked the time-related increase in th uptake. Depletion of glutamate did not lead to the enhanced uptake, whereas depletion of glycine and serine caused as much increase in the uptake as depletion of cystine did. The intracellular pool of glutathione was extremely reduced by depletion of cystine, or of glycine and serine, but to a far less extent by depletion of glutamate. The results indicate that te transport system for cystine and glutamate appears to undergo adaptive regulation. It is suggested that glutathione may function as a regulatory signal to this transport system.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.     

The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [(32)P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.     

The carbohydrate moieties bound to the carotenoids myxol and oscillol and their chemosystematic applications

The sugar moiety bound to myxol in Oscillatoria agardhii was shown by ¹H NMR (400 MHz) experiments and glycoside hydrolysis to be α-linked chinovose, tentatively with the L-configuration. Direct comparison of 100 MHz ¹H NMR spectra of the acetates of myxol α-chinovoside and of oscillaxanthin ex Arthrospira sp. suggests the same sugar component in oscillaxanthin, and also in myxoxanthophyll from the latter source. The O-methyl methylpentoside bound to myxol in O. bornetii f. tenuis was identified by ¹H NMR as α-linked 3-O-methyl-fucose, tentatively L-configurated. The differentiation of species in the genus Oscillatoria, causing natural blooms in eutrophic lakes, was supported by their carotenoid pattern when including the differences in sugar moiety of the carotenoid glycosides.