Isolation and characterization of human platelet glycoprotein IX

Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.     

Phosphorylation of the multidrug resistance associated glycoprotein

Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.     

Blood group N antigen precursor glycoproteins and N antigen precursor glycoproteins with Thomsen-Friedenreich (T) activity from human liver metastatic carcinomas.

1. Four perchloric acid-soluble fractions (PASFs) from human liver metastases of sigmoid colon carcinoma (LSCC), pancreas carcinoma (LPC), breast carcinoma (LBC) and human normal liver (NL) were subjected to DEAE-cellulose column chromatography and separated into three glycoprotein fractions, respectively. 2. In chromatography, LSCC-PASF and LBC-PASF gave Thomsen-Friedenreich (T)-active glycoprotein, blood group N antigen precursor glycoprotein with T activity and N antigen precursor glycoprotein, respectively. 3. LPC-PASF gave N antigen precursor glycoprotein with T activity and two N antigen precursor glycoproteins. 4. The three glycoprotein fractions from NL-PASF did not exhibit both T activity and N antigen precursor activity.     

Preparation and characterization of armadillo submandibular glycoproteins.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.     

Isolation of fatty acids covalently bound to the gastric mucus glycoprotein of normal and cystic fibrosis patients

Covalently bound fatty acids were found in strictly purified and delipidated gastric mucus glycoprotein of normal and cystic fibrosis individuals. The susceptibility of this linkage to methanolic KOH and hydroxylamine treatment indicated the ester bond between fatty acids and glycoprotein. On the average, 2.9 nmol fatty acid/mg glycoprotein were found in normal samples, and 12.2 nmol/mg glycoprotein in samples derived from cystic fibrosis. In normal gastric mucus glycoprotein the covalently linked fatty acids consisted of hexadecanoate (47.0%), octadecanoate (22.0%), tetracosanoate (5.9%), octadecenoate (14.5%) and tetracosenoate (6.0%). In cystic fibrosis mucus glycoprotein the covalently bound fatty acids were comprised mainly of hexadecanoate (36.5%), octadecanoate (48.7%) and octadecenoate (8.6%). These data indicate that cystic fibrosis gastric mucus glycoprotein is highly acylated and perhaps this is the major defect of glycoproteins in this disease.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

The synthesis and secretion of gastric mucus glycoprotein by mucosal cells cultured in the presence of ethanol

The effect of ethanol on the synthesis and secretion of mucus glycoprotein in gastric mucosal cells was investigated. The mucosal cell suspensions were subjected to a short-term (4 h) culture in the presence of 0-1.5 M ethanol, with [3H]proline and [3H]palmitic acid as markers for glycoprotein synthesis and acylation. The synthesized labeled mucus glycoprotein was isolated from the incubation medium (extracellular glycoprotein) and from the mucosal cells (intracellular glycoprotein), and analyzed. Depending upon the ethanol concentration in the cell culture medium, two distinct effects on the synthesis and secretion of mucus glycoprotein were observed. The cells cultured in the presence of 0.02-0.1 M ethanol showed increased ability for the incorporation of [3H]proline and [3H]palmitic acid, and for the secretion of the newly assembled mucus glycoprotein. The synthesis of the glycoprotein increased 18-fold, acylation 5-fold, and secretion 10-fold. The synthesized glycoprotein, however, contained four to five times less of acyl-bound fatty acids. Ethanol at 0.1-1.5 M caused a marked reduction (62-64%) in the mucus glycoprotein synthesis, but the amount of glycoprotein released to the medium remained constant. This indicated that higher concentrations of ethanol caused the release of the preformed intracellular mucus glycoprotein reserves. The results demonstrate that gastric mucosal cells incubated in the presence of ethanol exhibit impaired synthesis and secretion of mucus glycoprotein, and that the severity of impairment depends upon the ethanol concentration.     

Early cortical plate specific glycoprotein in a marsupial species belongs to the same family as fetuin and alpha 2HS glycoprotein

Two related glycoproteins, fetuin in species of the order Artiodactyla (cattle, sheep, pig) and alpha 2HS glycoprotein in the human [(1987) Cell Tissue Res. 248, 33-41] have a very specific distribution in the developing brain. We have isolated and determined the first 15 N-terminal residues of a similarly distributed glycoprotein in the developing brain of the tammar wallaby (Macropus eugenii). The degree of homology is the same between wallaby glycoprotein and alpha 2HS glycoprotein as between fetuin and alpha 2HS glycoprotein (46%). Antibodies made to synthetic peptides of fetuin were used to identify the wallaby glycoprotein. A polyclonal antibody to the purified glycoprotein was used for immunocytochemical identification of brain cells positive for this protein.     

Reassociation of membrane glycoprotein with intact erythrocytes

Purified membrane glycoprotein was found to specifically bind to erythrocyte surfaces. (1) The glycoprotein showed over 80-fold greater binding to erythrocytes than did bovine serum albumin; (2) the binding of the glycoprotein was not significantly inhibited by the presence of other proteins even when added in 175-fold greater concentration; (3) the glycoprotein's binding showed both time and concentration dependence. Radiolabeled glycoprotein was re-isolated after binding to erythrocytes and, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 64% of the label was recovered from the glycoprotein bands. The glycoprotein preparation would bind to erythrocyte surfaces to the extent of 6–7% of that naturally occurring in the membranes.     

Miltenberger Class I and II erythrocytes carry a variant of glycophorin A.

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.     

Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain.

Previous studies have shown that the glycoprotein cytoplasmic domains of human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus of macaques modulate biological activities of the viral glycoprotein complex, including syncytium formation, exterior glycoprotein conformation, and glycoprotein incorporation into budding virus particles. We have now utilized a recombinant expression system to study interactions of full-length or truncated HIV-2 glycoproteins with coexpressed HIV-2 Gag proteins which self-assemble and bud as virus-like particles. Interestingly, budding of HIV-2 virus-like particles from cells was enhanced 5- to 24-fold when Gag was coexpressed with the full-length HIV-2 glycoprotein, compared with Gag expressed either alone or with a truncated HIV-2 glycoprotein. The results obtained in this model system indicate that an additional effect of the lengthy cytoplasmic domain of the glycoprotein of HIV-2 is enhancement of particle budding. We speculate that the cytoplasmic domain of the viral glycoprotein of HIV-2 enhances budding by (i) potentiation of Gag structure or function or (ii) membrane modulation.     

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus.

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.     

Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein.

Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus.     

Properties of a glycopeptide isolated from human Tamm-Horsfall glycoprotein. Interaction with leucoagglutinin and anti-(human Tamm-Horsfall glycoprotein) antibodies.

A sialylated glycopeptide isolated after Pronase digestion of human Tamm-Horsfall glycoprotein behaves as a powerful monovalent hapten in the precipitin reaction between human Tamm-Horsfall glycoprotein and leucoagglutinin, but fails to inhibit the interaction of the glycoprotein with rabbit anti-(human Tamm-Horsfall glycoprotein) antibodies. The glycopeptide is much less active than the intact glycoprotein as an inhibitor of lymphocyte transformation induced by leucoagglutinin.     

The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus.

1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15 X 10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6 X 10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5 X 10(6), which is halved to 0.76 X 10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.     

The PO glycoprotein of peripheral nerve myelin

The PO glycoprotein, the major protein of peripheral nerve myelin, is a hydrophobic glycoprotein which can be isolated in soluble and insoluble forms from rabbit sciatic nerve myelin following extensive defatting and mid acidic extraction. The PO glycoprotein was localized exclusively in peripheral nervous system (PNS) myelin of sciatic nerve and rootlets by the immunofluorescent technique using goat anti-PO serum which showed a single precipitin band in double diffusion and did not cross-react with the myelin basic protein or P2 protein. Central nervous system (CNS) myelin from brain and spinal cord was negative by the immunofluorescent procedure. The major glycoprotein bands in PNS myelin, in addition to the PO glycoprotein at 28K, exist at 23K and 19K, as shown by gel electrophoresis in dodecyl sulfate. These glycoproteins, isolated by gel filtration in 2% dodecyl sulfate, show identity to the PO glycoprotein in their monosaccharide profile and overlapping tryptic peptides on peptide mapping. We conclude that both the 23K and 19K glycoproteins are derived from the PO glycoprotein by in situ proteolysis; the 23K glycoprotein has the identical amino terminal sequence. The 19K glycoprotein, beginning with amino-terminal methionine, is identical with the TPO glycoprotein, shown previously to originate from tryptic hydrolysis of the PO glycoprotein in isolated myelin. A tryptic glycopeptide containing 27 amino acids was isolated from the PO glycoprotein and sequenced. It contained a relatively high proportion of aspartic acid (four residues) and glutamic acid (two residues), thus exhibiting a high negative charge. We conclude that the total carbohydrate of the PO, 23K, and 19K glycoproteins does indeed exist as a single nonasaccharide moiety linked through N-acetylglucosamine to Asp-14 of the glycopeptide in a N-glycosidic linkage. These results further support the role of the PO glycoprotein as a typical amphipathic membrane protein.     

Hypolipidemic effect and antioxidant activity of glycoprotein isolated from Ulmus davidiana Nakai in Triton WR-1339-treated mouse

The glycoprotein isolated from Ulmus davidiana Nakai (UDN) (UDN glycoprotein) has a molecular weight of 116 kDa and consists of 78.65% carbohydrate content and 21.35% protein content. In the present study, we investigated the hypolipidemic effect of UDN glycoprotein on Triton WR-1339-induced mice. With pretreatment with UDN glycoprotein, the triacylglycerol (TAG), total cholesterol and low density lipoprotein-cholesterol (LDL-C) concentrations were significantly reduced, whereas high density lipoprotein-cholesterol (HDL-C) concentration was increased in the plasma of Triton WR-1339-induced mice. With respect to antioxidative activity, UDN glycoprotein significantly decreased the level of thiobarbituric acid reactive substances (TBARS) and improved activities of catalase and glutathione peroxidase (GPx), without an apparent change of superoxide dismutase (SOD) activity. Also UDN glycoprotein significantly increased nitric oxide (NO) production in Triton WR-1339-induced mice. These results indicate that UDN glycoprotein has a hypolipidemic effect, possesses antioxidant activity and has an ability to stimulate NO production. Thus, we speculate that UDN glycoprotein is an example of natural compound that lowers plasma lipid level together with having an antioxidant function in Triton WR-1339-induced mice.     

Thomsen-Friedenreich (T)-active glycoproteins, blood group N antigen precursor glycoproteins and N antigen precursor glycoproteins with T activity from ascitic fluids of liver cancer patients.

1. Five perchloric acid-soluble fractions (PASFs) obtained from ascitic fluids of three patients with primary hepatocellular carcinoma (HCC), a patient with liver metastatic carcinoma (LUC) from ureteral carcinoma and human normal serum (NS) were subjected to DEAE-cellulose column chromatography to separate seven glycoprotein fractions, respectively. 2. In this chromatography, two HCC-PASFs gave a Thomsen-Friedenreich (T)-active glycoprotein, respectively. 3. Other HCC-PASF gave a T-active glycoprotein, two blood group N antigen precursor glycoproteins and an N antigen precursor glycoprotein with T activity. 4. LUC-PASF gave two T-active glycoproteins and an N antigen precursor glycoprotein with T activity. 5. NS-PASF did not give these serologically active glycoproteins.     

Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein.

In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions of strains that do not express HE, such as MHV-A59, can infect mouse fibroblasts in vitro, showing that the HE glycoprotein is not required for infection of these cells. The present work was done to study whether interaction of the HE glycoprotein with carbohydrate moieties could lead to virus entry and infection in the absence of interaction of the S glycoprotein with its receptor glycoprotein, MHVR. The DVIM strain of MHV expresses large amounts of HE glycoprotein, as shown by hemadsorption, acetylesterase activity, and immunoreactivity with antibodies directed against the HE glycoprotein of bovine coronavirus. A monoclonal anti-MHVR antibody, MAb-CC1, blocks binding of virus S glycoprotein to MHVR and blocks infection of MHV strains that do not express HE. MAb-CC1 also prevented MHV-DVIM infection of mouse DBT cells and primary mouse glial cell cultures. Although MDCK-I cells express O-acetylated sialic acid residues on their plasma membranes, these canine cells were resistant to infection with MHV-A59 and MHV-DVIM. Transfection of MDCK-I cells with MHVR cDNA made them susceptible to infection with MHV-A59 and MHV-DVIM. Thus, the HE glycoprotein of an MHV strain did not lead to infection of cultured murine neural cells or of nonmurine cells that express the carbohydrate ligand of the HE glycoprotein. Therefore, interaction of the spike glycoprotein of MHV with its carcinoembryonic antigen-related receptor glycoprotein is required for infectivity of MHV strains whether or not they express the HE glycoprotein.