Molecular Weight Glycoprotein Research Articles

Isolation and partial characterization of a soluble mucin in human pancreatic juice

Morphological and histochemical abnormalities in pancreatic mucin occur in many pancreatic disorders. However, the composition of pancreatic mucin is poorly understood. Purified mucin was isolated from pure pancreatic juice by sequential chromatography on Sepharose CL-2B and CL-4B followed by CsCl density gradient ultracentrifugation. The mucin preparation consists of 24% protein and 73% carbohydrate. Reduction of the macromolecule (greater than 2 x 10(6)) by mercaptoethanol resulted in the formation of subunits of molecular weight 500,000 and released several small molecular weight proteins, including a glycoprotein of an average molecular weight of 116,000. Cellulose acetate electrophoresis separated the mucin into three species of different staining properties for periodic acid-Schiff reagent and Alcian blue, suggesting the presence of microheterogeneity with respect to sulphation and sialation. Threonine, serine, and proline composed 48% of the total amino acids, while the oligosaccharide moiety contained N-acetylglucosamine, N-acetylgalactosamine, fucose, galactose, sialic acid, and sulphate. We also detected the presence of C16:0 and C18:0 fatty acids which were probably noncovalently bound to the pancreatic mucin.

Preparation of Pancreatic Cancer-Associated Mucin Expressing CA19–9, CA50, Span-1, Sialyl SSEA-1, and Dupan-2

A new antigen associated with pancreatic cancer was prepared by immunoaffinity chromatography using Fab'-Sepharose beads. This antigen was a glycoprotein of large molecular weight (Mr greater than 8,000,000) in its native state, estimated by size exclusion chromatography on Sephacryl S400. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting analysis, several cancer-associated glycoconjugates, including CA19-9, CA50, Span-1, Dupan-2, and sialyl SSEA-1, were detected on the antigenic moiety of Mr 90,000. By an enzyme immunoassay for the antigen, elevated levels were found in pooled sera obtained from patients with various malignant and non-malignant diseases and normal subjects. However, the enhanced expression of CA19-9, Lewisa, or Lewisb epitope on the antigen molecule was restricted to the pooled sera from patients with pancreatic cancer. Furthermore, antigens from pancreatic or gastric cancer expressed ligands with intense and specific reactivity for Bauhinia purpurea (BPA), peanut (PNA), and Vicia villosa (VVA) lectins. The present assay system of the antigen, using both monoclonal antibodies (CA19-9, Lewisa, and Lewisb) and lectins (BPA, VVA and PNA), will provide a useful approach to the diagnosis of pancreatic cancer.

Seminal Plasma Protein p30: Simplified Purification and Evidence for Identity with Prostate Specific Antigen

A rapid high yield purification scheme is described for the major 30,000 molecular weight glycoprotein (p30) in human seminal plasma. Immunological and protein sequence evidence demonstrates the identity of this protein with prostate specific antigen (PSA) and gamma-seminoprotein (γ SM).

Analysis of human antibody responses to human cytomegalovirus envelope glycoproteins found in two families of disulfide linked glycoprotein complexes designated gC-I and gC-II

Human antibody responses to human cytomegalovirus (HCMV) envelope glycoproteins were analyzed using immunoaffinity purified glycoproteins and Western blotting. Two families of disulfide linked glycoprotein complexes, designated gC-I and gC-II, were isolated. These complexes were reduced and their individual glycoproteins separated by polyacrylamide gel electrophoresis prior to electroblotting. The reactivity of adult convalescent sera with individual glycoproteins was compared to that of sera from congenitally infected infants. All sera tested reacted with a 52,000 molecular weight glycoprotein from these complexes, but only 75% reacted with a 93,000 to 130,000 molecular weight glycoprotein from gC-I complexes. Most adult convalescent sera reacted with glycoproteins from gC-II complexes. However, 14 of 16 infant sera did not have high enough levels of gC-II antibodies to give a positive reaction with Western blotting. A longitudinal study was done with several infants and their mothers. These studies indicated a failure by the infants and their mothers to develop detectable levels of gC-II antibodies months to years after the initial infection or after repeated stimulation with HCMV due to persistent infection. The inability of these infants to develop significant levels of gC-II antibodies was not due to an inability to respond to viral glycoproteins since they had antibodies to gC-I glycoproteins. We also determined that the strains of HCMV infecting some of these infants expressed gC-II glycoproteins. Thus, the lack of response by these infants was not due to lack of expression of gC-II glycoproteins by their infecting strain.

Physical mapping, amplification, and overexpression of the mouse mdr gene family in multidrug-resistant cells.

The mouse mdr gene family consists of three distinct genes (mdr1, mdr2, and mdr3), for which we have isolated full-length cDNA clones. cDNA subfragments corresponding to discrete regions showing little sequence conservation among the three mdr genes were used as gene-specific DNA probes in hybridization experiments. Long-range mapping by pulse-field gel electrophoresis indicated that the three mdr genes are closely linked on a genomic DNA segment of approximately 625 kilobases. The gene order and direction of transcription of the three genes were determined and indicate the arrangement (5') mdr3 (3')-(5') mdr1 (3')-(3') mdr2 (5'). Southern blotting analyses of genomic DNA from a panel of independently derived multidrug-resistant cell lines identified mdr gene amplification in 10 of 12 cell lines studied. In individual cell lines showing gene amplification, the copy number of each of the three mdr genes was identical, suggesting that the three mdr genes became amplified as part of a single amplicon in these cells. Although increased expression of all three mdr genes was detected in 2 of 12 cell lines tested, multidrug resistance was associated in 10 of 12 lines with the independent overexpression of either mdr1 (7 of 12) or mdr3 (3 of 12) but not mdr2. mdr1 overexpression was consistently associated with gene amplification, while increased mdr3 expression was detected in certain cell lines that did not show gene amplification. Increased levels of mdr1 mRNA were linked to the overexpression of a P glycoprotein of apparent molecular weight 180,000 to 200,000, whereas increased mdr3 expression resulted in increased expression of a P glycoprotein of molecular weight 160,000 to 180,000. Our results suggest that at least two members of the mouse mdr gene family, mdr1 and mdr3, can independently confer multidrug resistance in the cell lines examined.

Physical Mapping, Amplification, and Overexpression of the Mouse mdr Gene Family in Multidrug-Resistant Cells

The mouse mdr gene family consists of three distinct genes (mdr1, mdr2, and mdr3), for which we have isolated full-length cDNA clones. cDNA subfragments corresponding to discrete regions showing little sequence conservation among the three mdr genes were used as gene-specific DNA probes in hybridization experiments. Long-range mapping by pulse-field gel electrophoresis indicated that the three mdr genes are closely linked on a genomic DNA segment of approximately 625 kilobases. The gene order and direction of transcription of the three genes were determined and indicate the arrangement (5') mdr3 (3')-(5') mdr1 (3')-(3') mdr2 (5'). Southern blotting analyses of genomic DNA from a panel of independently derived multidrug-resistant cell lines identified mdr gene amplification in 10 of 12 cell lines studied. In individual cell lines showing gene amplification, the copy number of each of the three mdr genes was identical, suggesting that the three mdr genes became amplified as part of a single amplicon in these cells. Although increased expression of all three mdr genes was detected in 2 of 12 cell lines tested, multidrug resistance was associated in 10 of 12 lines with the independent overexpression of either mdr1 (7 of 12) or mdr3 (3 of 12) but not mdr2. mdr1 overexpression was consistently associated with gene amplification, while increased mdr3 expression was detected in certain cell lines that did not show gene amplification. Increased levels of mdr1 mRNA were linked to the overexpression of a P glycoprotein of apparent molecular weight 180,000 to 200,000, whereas increased mdr3 expression resulted in increased expression of a P glycoprotein of molecular weight 160,000 to 180,000. Our results suggest that at least two members of the mouse mdr gene family, mdr1 and mdr3, can independently confer multidrug resistance in the cell lines examined.

Expression of c-erbB-2 Protein Detected in Adenocarcinoma Arising from Parotid Pleomorphic Adenoma

The c-erbB-2 proto-oncogene is known to encode a 185,000 molecular weight glycoprotein. This protein has been detected immunohistochemically in several human adenocarcinomas, suggesting that it may play a role in the development of these malignancies. In the otolaryngological field none of the adenocarcinomas expressing c-erbB-2 protein has yet been described. In this article we presented a case of parotid adenocarcinoma expressing the c-erbB-2 protein. In this case the adenocarcinoma was thought to have originated from pleomorphic adenoma. Immunohistochemically, the adenocarcinoma cells were stained and the remaining pleomorphic adenoma cells were not stained by polyclonal antibody against the c-erbB-2 protein. The expression of c-erbB-2 protein may have been related to the malignant development of the pleomorphic adenoma.

Isolation and Characterization of a Novel Large Protease Accumulated in Mammalian Cells in the Presence of Inhibitors

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.

Development of theAedes aegyptiChorion: Proteins and Ultrastructure

Summary The appearance of discrete proteins in the Aedes aegypti chorion (eggshell), prepared from the ovaries at different stages by a gentle lysis-washing method, may be correlated with the developing chorionic ultrastructure. Scanning and transmission electron microscopy and SDS-gel electrophoresis indicate that the smooth endochorion is assembled first on the oocyte's outer membrane from several 90,000 and ca. 18,000 molecular weight proteins. The tubercle-patterned exochorion is then assembled from a group of 25,000-50,000 molecular weight glycoproteins, probably chitin, and several very high molecular weight proteoglycans.

Purification and characterization of allergens from Xanthium strumarium pollen.

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic components. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103,000 daltons. Xan VIa was a glycoprotein of molecular weight 17,000 daltons. The carbohydrate moiety of Xan VIa was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.

Production of a monoclonal antibody specific for seminomas and dysgerminomas.

A monoclonal antibody (M2A, IgG2a) was produced against a cultured human ovarian epithelial adenocarcinoma cell line, HEY. Monoclonal antibody M2A reacted with a glycoprotein of molecular weight 40,000 on the surface of HEY cells. The affinity constant of the monoclonal antibody M2A for HEY cells was 10(9) M-1, and the number of binding sites on HEY cells was 2 X 10(4) per cell. The monoclonal antibody produced positive immunoperoxidase staining of fetal (but not adult) testis and of seminomas and dysgerminomas but did not stain various normal adult tissues or other gonadal or extragonadal tumors. Monoclonal antibody M2A may be useful for confirming a histological diagnosis of seminoma and dysgerminoma.

Development of Monoclonal Antibodies to the Human Breast Carcinoma Cell Line PMC42<xref ref-type="fn" rid="FN1">2</xref>

In an attempt to identify antigens expressed during breast differentiation, three murine monoclonal antibodies, CIBr2, CIBr7, and CIBr18, were produced against the human pleomorphic breast carcinoma cell line PMC42. All three monoclonal antibodies reacted with previously undescribed antigenic determinants on the PMC42 cell line. Antibody CIBr18 reacted only with the immunizing cell line PMC42, whereas antibodies CIBr2 and CIBr7 showed minimal reactivity toward a panel of 34 human leukemia- and solid tumor-derived cell lines. The antigenic determinants detected by the three antibodies were distinct, and each showed variable expression in PMC42 monolayer and organoid cultures. The heterogeneity of staining seen on PMC42 cultures may reflect the fact that this cell line contains up to eight morphologically distinct cell types. Antigen expression correlated with cell type in some instances, whereas in other instances phenotypic subdivision within a cell type was apparent. Antigens recognized by antibodies CIBr7 and CIBr18 were characterized biochemically. In Western blotting, antibody CIBr7 identified a single band of an apparent molecular weight of 38,000 within PMC42 cell lysates. Sodium dodecyl sulfate-polyacrylamide gel analysis of polypeptides immunoprecipitated by antibody CIBr18 from [35S]methionine-labeled PMC42 cell lysates identified two glycoproteins of apparent molecular weights of 115,000 and 120,000, respectively. No biochemical data for the CIBr2 antigen are yet available. All three antigens were detected in human mammary epithelium and some non-breast tissues. The expression of these antigens in normal and neoplastic mammary epithelia is discussed in terms of antigen heterogeneity and changes in antigen expression upon conversion to the malignant state.

An intrinsic membrane glycoprotein of the lens.

A major glycoprotein of relative molecular weight of 130,000 daltons of chick lens cell membranes was identified by Concanavalin-A staining and enriched for by Con A-Sepharose chromatography. Using both Con A binding and immunological analyses with a specific polyclonal antibody, the glycoprotein was detected in chick lens epithelium, annular pad, cortex and nucleus. Localization to the plasma membranes of lens cells was demonstrated by immunofluorescence. A similar protein, which cross-reacts with the antibody to the chick protein and also binds Con A was demonstrated in human and bovine lenses.

The chemokinetic inhibitory factor (CIF) in serum of CLL patients: correlation with infection propensity and disease activity.

We have recently described the partial purification and characterization of a neutrophil migration inhibitory activity present in serum from patients with chronic lymphocytic leukaemia (CLL). This new lymphokine, the chemokinetic inhibitory factor (CIF), is produced by B-CLL cells. It is a heat-labile glycoprotein of an approximate molecular weight (m. w.) of 30000. In this extended investigation 64/89 CLL-patients had CIF in their serum. CLL serum diluted to a concentration of 0.02% gave significantly decreased chemokinetic activity, suggesting that CIF is potent at very low concentrations. 31/89 patients had increased infection propensity. Significantly more patients with CIF in serum had infections compared to the group with normal susceptibility to infections. The combination of low Ig levels and CIF in serum discriminated even better between the infection-prone and non-infection-prone patients. CIF in serum was not correlated to tumour cell mass - estimated by Rai clinical staging - tumour progression or deoxythymidine kinase, S-TK, an enzyme that may reflect proliferating cells. The existence of this new lymphokine in serum seems to contribute to the increased susceptibility to infections seen in CLL patients.

The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin.

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.

Properties of rat striatal D-2 dopamine receptors solubilized with the zwitterionic detergent CHAPS.

The specific binding of [3H]spiperone was determined in membrane preparations of rat striatum and following solubilization treatment with the zwitterionic detergent CHAPS. Membrane protein solubilization was confirmed by ultrafiltration, gel filtration and increased heat sensitivity. Specific binding of [3H]spiperone to the solubilized preparation was saturable and of high affinity, although solubilization led to an approximate 10 fold decrease in receptor affinity for [3H]spiperone. The drug displacement profile of binding to the CHAPS solubilized preparation corresponded to that of the dopamine D-2 receptor; binding was stereoselectively displaced by the isomers of butaclamol. The sodium dependence of sulpiride displacement of specific [3H]spiperone binding was retained in the CHAPS solubilized preparation. GTP (100 microM) only altered the ability of dopamine to displace [3H]spiperone binding to the solubilized preparation in the presence of 120 mM sodium chloride. The GTP effect was small compared with that observed in the membranes. Specific [3H]spiperone binding sites in the solubilized preparation were preferentially retained by a wheat germ agglutinin affinity column and subsequently eluted with N-acetyl-D-glucosamine. Gel filtration of the solubilized preparation using a Sepharose column resulted in two peaks of specific [3H]spiperone binding, the larger component had a Stokes radius of 7.7 nm. CHAPS treatment of rat striatal membranes results in solubilization of the D-2 receptor in an active form. The D-2 site appears to be a glycoprotein of high molecular weight.

Receptors and recognition mechanisms of Trypanosoma cruzi

The current state of knowledge on receptor and recognition interactions which take place during the life-cycle of Trypanosoma cruzi is reviewed. Evidence suggests that carbohydrate plays a central role in these recognition mechanisms. Lectin-sugar interactions appear to be involved in uptake of the parasite by host cells including macrophages and a protein on the surface of trypomastigote which binds N-acetyl glucosamine on the host cell has been implicated in host cell invasion. Sugars on a 72,000 molecular weight glycoprotein on epimastigotes have also been implicated in colonization of the gut of the insect vector and in control of the morphological changes which take place in the insect gut.

Further evaluation of lectin affinity purified glycoprotein (GP90) in the enzyme linked immunosorbent assay (ELISA) for diagnosis of Trypanosoma cruzi infection

Sera from 143 patients considered to be infected with Trypanosoma cruzi on the basis of epidemiological, clinical and standard serological evidence gave positive results in the enzyme-linked immunosorbent assay (ELISA) using a lectin affinity purified 90,000 molecular weight glycoprotein (GP90) antigen preparation. Levels of antibody did not discriminate between clinically classified groups of patients in the chronic phase of infection. The GP90 preparation was found to be heterogeneous.

Fibronectin-containing hyaline globules in malignant melanoma

Hyaline globular inclusions were found in a case of malignant melanoma. The globules were both intracellular and extracellular in the primary neoplasm and in metastases to lymph nodes, liver, and lung. They were studied immunohistochemically and were found to contain fibronectin, a glycoprotein of high molecular weight. Ultrastructurally, the globules were composed of electron-dense, finely fibrillar aggregates. It is suggested that the neoplastic cells produced the fibronectin, which accumulated in globular form. Ten additional cases of malignant melanoma were studied; none of these had hyaline globules or intracellular globular fibronectin. The globules were compared to similar structures described in Spitz's nevi and other neoplasms. Immunohistochemical analysis was most useful in distinguishing the globules found in the malignant melanoma from those found in other conditions.

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.