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PubMed1996

Spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection.

The mouse hepatitis virus (MHV) spike glycoprotein mediates attachment of the virus to the MHV receptor, the murine biliary glycoprotein (BGP) carcinoembryonic antigen. Monoclonal antibody CC1 directed against BGP specifically inhibited infection of DBT, Sac-, GT1-7, and OBL21 cells by wild-type MHV-4 and the neuron-adapted variant OBLV60. Binding to this receptor was necessary to establish infection by cell-free MHV; however, the presence of BGP was not required for infection by cell-associated virus. Cell-associated infectious induced syncytium formation on Vero and BHK cells, which lack murine BGP; this activity was not inhibited by monoclonal antibody CC1. Antibody CC1 also did not prevent syncytium formation on DBT cells, which bear BGP. In infectious center assays, the MHV-4 variant OBLV60, which exhibits acid-dependent fusion, spread to cells lacking BGP only when exposed to acidic media. Therefore, spike-mediated fusion was required for BGP-independent spread of MHV infection. Furthermore, BGP-independent, cell-associated spread of MHV-4 was prevented by monoclonal antibodies 5A13.5 and 5B19.2 directed against the spike glycoprotein, but not by other neutralizing and nonneutralizing anti-spike antibodies. Expression of spike glycoprotein by recombinant vaccinia virus resulted in fusion of BGP-negative cells; monoclonal antibodies 5A13.5 and 5B19.2 strongly inhibited spike-mediated fusion in this assay.

Animals↗
PubMed1999

A 42-kDa glycoprotein from chicken egg-envelope, an avian homolog of the ZPC family glycoproteins in mammalian Zona pellucida. Its first identification, cDNA cloning and granulosa cell-specific expression.

A glycoprotein with molecular mass of 42 kDa was identified as the major component of the chicken egg-envelope, the filamentous, extracellular matrix known as the perivitelline layer. By using a DNA probe amplified with degenerative primers derived from the protein's partial amino acid sequences, a cDNA clone encoding the egg-envelope 42-kDa glycoprotein (gp42) was isolated from a hen's ovary cDNA library. The gp42 open reading frame encoded 435 amino acid residues, including a putative signal peptide of 20 amino acids. The deduced amino acid sequence of gp42 showed significant similarity to egg-envelope glycoproteins of the ZPC family of several other vertebrate species, including human ZP3, mouse ZP3, Xenopus laevis gp43 and medaka (Oryzias latipes) ZI3 (LS-F), which play important roles for sperm-egg interaction. A single N-glycosylation site present in chicken gp42 is conserved among all five of these proteins: carbohydrate analysis of gp42 revealed the presence of a complex type glycan chain at this site. N-terminal sequence analysis of the mature polypeptide suggests that C-terminal processing of the pro-protein occurs during synthesis and secretion. The 1.4-kb gp42 transcript was detected only in follicles, and was found to be accumulated in granulosa cells in a manner dependent on ovarian follicular development. Furthermore, a metabolically radio-labeled gp42 was immunopreciptated from both cell lysate and culture supernatant of the granulosa cells with specific anti-gp42 antibody, suggesting granulosa cell-specific synthesis and secretion of the glycoprotein.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a bovine herpesvirus-1 (BHV-1) glycoprotein gL which is required for proper antigenicity, processing, and transport of BHV-1 glycoprotein gH.

DNA sequence analysis of the bovine herpesvirus-1 (BHV-1) genome revealed the presence of an open reading frame named UL1 which exhibited limited homology to glycoprotein gL of herpes simplex virus-1 (S. K. Khattar, S. van Drunen Littel-van den Hurk, L. A. Babiuk, and S. K. Tikoo, Virology 213, 28-37). To identify the BHV-1 UL1 protein, rabbit antisera were prepared against two synthetic peptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides immunoprecipitated a 16- to 17-kDa protein from in vitro translated in vitro transcribed mRNA, BHV-1-infected MDBK cells, and purified virions. Enzymatic deglycosylation and lectin binding assays confirmed that the BHV-1 UL1 protein contains only O-linked oligosaccharides and was named glycoprotein gL. Sera against UL22 protein immunoprecipitated a protein of 108 kDa from BHV-1-infected MDBK cells and purified virions, which was modified only by N-linked oligosaccharides and was named glycoprotein gH. Glycoprotein gL expressed by recombinant vaccinia virus was properly processed and secreted into the medium. In contrast glycoprotein gH expressed by recombinant vaccinia virus was found to be retained in the rough endoplasmic reticulum. However, gH coexpressed with gL by recombinant vaccinia viruses was properly processed and transported to the cell surface, suggesting that complex formation between gH and gL is necessary for the proper processing and transport of gH but not gL. In addition gH--gL complex formation is also required for induction of neutralizing antibody response and anchoring of gL to the plasma membrane.

Animals↗
PubMed2000

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.

3T3 Cells↗
PubMed1985

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC.

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.

Animals↗
PubMed1991

Liquid chromatographic method for the determination of the carbohydrate moiety of glycoproteins. Application to alpha 1-acid glycoprotein and tissue plasminogen activator.

A rapid procedure is described for the qualitative and quantitative analysis of the carbohydrate composition of glycoproteins by liquid chromatography with light-scattering detection. The analysis was carried out in three steps. First, the glycoprotein samples were purified by a two-step purification on a Sephadex G-25 column with a 90% yield. Second, the selectivity of the separation and the sensitivity of detection of monosaccharides, as methyl glycosides obtained by direct methanolysis of glycoproteins, were improved by modified simplex optimization of the methanolysis parameters (temperature, methanolic hydrochloric acid strength and reaction time) determined at 66 degrees C, 1.2 M and 8.1 h for alpha 1-acid glycoprotein (alpha-AGP) and 73 degrees C, 1.5 M and 12.5 h for tissue plasminogen activator (tPA). Finally, the method was applied to the determination of the carbohydrate moiety of the two N-glycosylated glycoproteins alpha-AGP and tPA.

Acetylglucosamine↗
PubMed1999

Role of P-glycoprotein-mediated secretion in absorptive drug permeability: An approach using passive membrane permeability and affinity to P-glycoprotein.

It has been shown in vivo and in vitro that P-glycoprotein (P-gp) may be able to influence the permeability of its substrates across biological membranes. However, the quantitative contribution of the secretion process mediated by P-gp on the overall permeability of membranes has not been determined yet. In particular, observations need to be clarified in which substrates showing high affinity to P-glycoprotein, e.g., verapamil, apparently do not seem to be greatly influenced by P-gp in their permeability and consequently also with respect to their extent of GI-absorption after oral administration, whereas weaker substrates of P-gp, e.g., talinolol, have clearly shown P-gp-related absorption phenomena such as nonlinear intestinal permeability and bioavailability. Experiments with Caco-2 cell monolayers and mathematical simulations based on a mechanistic permeation model should aid in clarifying the underlying mechanism for these observations and quantifying the influence of passive membrane permeability and affinity to P-gp to the overall transmembrane drug flux. In addition, the concentration range of drug at which P-glycoprotein-mediated transport across the biological membrane is relevant should be examined. The permeability of various drugs in Caco-2 monolayers was determined experimentally and modeled using a combination of passive absorptive membrane permeability and a Michaelis-Menten-type transport process in the secretory direction. The passive permeabilities were experimentally obtained for the apical and basolateral membrane by efflux experiments using Caco-2 monolayers in the presence of a P-gp inhibitor. The Michaelis-Menten parameters were determined by a newly developed radioligand-binding assay for the quantification of drug affinity to P-gp. The model was able to accurately simulate the permeability of P-glycoprotein substrates, with differing passive membrane permeabilities and P-glycoprotein affinities. Using the outlined approach, permeability vs donor-concentration profiles were calculated, and the relative contribution of passive and active transport processes to the overall membrane permeability was evaluated. A model is presented to quantitatively describe and predict direction-dependent drug fluxes in Caco-2 monolayers by knowing the affinity of a compound to the exsorptive transporter P-gp and its passive membrane permeability. It was shown that a combination of high P-gp affinity with good passive membrane permeability, e.g., in the case of verapamil, will readily compensate for the P-gp-mediated reduction of intestinal permeability, resulting in a narrow range in which the permeability depends on the apical drug concentration. On the other hand, the permeability of compounds with low passive membrane permeability (e. g., talinolol) might be affected over a wide concentration range despite low affinity to P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Stimulation of glycoprotein gp120 dissociation from the envelope glycoprotein complex of human immunodeficiency virus type 1 by soluble CD4 and CD4 peptide derivatives: implications for the role of the complementarity-determining region 3-like region in membrane fusion.

We have used a recombinant vaccinia virus vector encoding the envelope glycoprotein of human immunodeficiency virus type 1 to study receptor-induced structural changes related to membrane fusion. A truncated soluble form of human CD4 (sCD4) was found to stimulate dissociation of the external subunit (gp120) from the envelope glycoprotein complex of human immunodeficiency virus type 1 expressed at the cell surface. sCD4 stimulation of gp120 release was time- and concentration-dependent and was associated with specific binding of sCD4 to gp120. Synthetic peptide derivatives corresponding to residues 81-92 of human CD4 (overlapping the complementarity-determining region 3-like region) inhibited cell-cell fusion mediated by the interaction between recombinant vaccinia-encoded CD4 and human immunodeficiency virus envelope glycoprotein. These peptide derivatives also stimulated gp120 release from the envelope glycoprotein complex. An analogous peptide derivative from chimpanzee CD4 (containing a single Glu----Gly substitution at the position corresponding to CD4 residue 87) was considerably less active at inhibition of cell-cell fusion and stimulation of gp120 release, consistent with the known inhibitory effect of this substitution on the ability of membrane-associated CD4 to mediate cell fusion. These results suggest that the sCD4-induced release of gp120 reflects postbinding structural changes in the envelope glycoprotein complex involved in membrane fusion, with the complementarity-determining region 3-like region playing a critical role.

CD4 Antigens↗
PubMed1988

Glycoprotein processing and glycoprotein processing inhibitors.

Considerable evidence is now accumulating from both in vivo and in vitro studies that the oligosaccharide chains of the plant N-linked glycoproteins undergo modification or processing reactions after the oligosaccharide has been transferred from its lipid-linked oligosaccharide intermediate to the protein. These processing reactions occur in the endoplasmic reticulum and Golgi apparatus of the cell and involve the removal of certain sugars and the addition of other sugars. While the processing reactions appear to be generally similar to those that occur in animal cells, there are some notable differences, such as the addition of a beta-linked xylose to many of the plant glycoproteins. It will be interesting to determine the exact sequence of these reactions and how they are regulated in the cell. Recently, some very useful inhibitors have become available that act on the glycosidases that catalyze the removal of glucose and mannose. These inhibitors cause the accumulation of aberrant oligosaccharide chains on the glycoproteins. Such unusual glycoproteins should be valuable tools for studies on the role of oligosaccharide in glycoprotein function.

Journal Article↗
PubMed1988

Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gp13) with homology to herpes simplex virus glycoprotein C.

The molecular structure of the equine herpesvirus type 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was analyzed. The gene is contained within a 1.8-kilobase AccI-EcoRI restriction fragment mapping at map coordinates 0.136 to 0.148 in the UL region of the EHV-1 genome and is transcribed from right to left. Determination of the nucleotide sequence of the DNA fragment revealed a complete transcriptional unit composed of typical regulatory promoter elements upstream to a long open reading frame (1,404 base pairs) that encoded a 468-amino-acid primary translation product of 51 kilodaltons. The predicted protein has the characteristic features of a membrane-spanning protein: an N-terminal signal sequence, a hydrophobic membrane anchor region, a charged C-terminal cytoplasmic tail, and an exterior domain with nine potential N-glycosylation sites. The EHV-1 DNA sequences expressed in lambda gt11 as gp13 epitopes were present in the open reading frame. Amino acid sequences composing a major antigenic site, recognized by 35% of a panel of 42 anti-gp13 monoclonal antibodies, were identified in the N-terminal surface domain of the deduced gp13 molecule. Comparison of the EHV-1 gp13 DNA sequence with that encoding glycoproteins of other alphaherpesviruses revealed no detectable homology. However, a search for homology at the amino acid level showed regions of significant sequence similarity between the amino acids of the carboxy half of EHV-1 gp13 and those of the same region of gC-like glycoproteins of herpes simplex virus (gC-1 and gC-2), pseudorabies herpesvirus (gIII), and varicella-zoster virus (gp66). The sequences of the N-terminal portion of gp13, by contrast, were much less conserved. The results of these studies indicate that EHV-1 gp13 is the structural homolog of herpes simplex virus glycoprotein C and further suggest that the epitope-containing N-terminal amino acid sequences of the herpesvirus gC-like glycoproteins have undergone more extensive evolutionary divergence than the C-terminal sequences.

Amino Acid Sequence↗
PubMed1991

Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is integrated into the PrV envelope and can functionally replace glycoprotein gII of PrV.

Animals↗
PubMed1991

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.

Amino Acid Sequence↗
PubMed1998

Stabilization of human immunodeficiency virus type 1 envelope glycoprotein trimers by disulfide bonds introduced into the gp41 glycoprotein ectodomain.

Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.

Amino Acid Sequence↗
PubMed1990

Vaccination against pseudorabies with glycoprotein gI+ or glycoprotein gI- vaccine.

Subunit pseudorabies vaccines that contained only purified glycoproteins of either of 2 strains of pseudorabies virus (PRV) were prepared and subsequently tested for safety and efficacy. The strains of virus used for vaccine production differed in at least 2 properties. One strain (Kojnok) was virulent for pigs and was believed to code for the entire complement of viral glycoproteins. The other (Kaplan) was a deletion mutant that was unable to code for structural viral glycoproteins gI and gp63. Purified glycoproteins were dispersed in an oil-in-water emulsion and were administered IM to pigs. Both vaccines were found to be safe and effective immunogens. Neither caused any local or general reactions, as verified by examination of the injection site (local safety) and by vaccination of pregnant sows in PRV-infected and noninfected herds. Sows vaccinated with the gI+ or gI- vaccine protected their pigs at levels of 93 and 92%, respectively, against a severe challenge exposure that killed 98% of pigs born from nonvaccinated sows. Vaccinated pigs were tested for active immunity by intranasal challenge exposure with the NIA 3 strain. Protection was quantitated by measuring the relative daily weight difference, expressed in percent per day, between vaccinated and control pigs during the first week after challenge exposure (delta G7); the estimated differences were 2.25 and 2.13% for gI+ and gI- vaccines, respectively. The absence of gI and gp63 did not affect the efficacy of this type of subunit glycoprotein vaccines.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The major 40-kDa glycoprotein in human prostatic fluid is identical to Zn-alpha 2-glycoprotein.

A major 40-KDA protein secreted by human prostate was isolated from whole seminal plasma by sequential column chromatography on DEAE-Sepharose CL-6B, concanavalin A(Con A)-Sepharose, and Sephadex G-100. Although the purified preparation still contained minor contaminants, its amino acid composition was found to be identical to the one of a glycoprotein isolated previously from seminal plasma by Lin et al (1983). Antibodies against this protein were produced in rabbits and their use in immunoblotting experiments revealed the presence of the antigen in several tissues including the prostate, the liver, the heart, the kidney, the epididymis, and the testis. A radioimmunoassay confirmed these results and showed that blood serum concentrations of this protein were relatively high in men (81 +/- 3 micrograms/ml), women (68 +/- 3 micrograms/ml), and cord blood of newborns (32 +/- 1 micrograms/ml). The serum concentrations of this protein along with its physicochemical characteristics suggested that it could be identical to Zn-alpha 2-glycoprotein, a human serum protein previously isolated by Burgi and Schmid (1961). This hypothesis was confirmed by a double immunodiffusion analysis using a commercial anti-Zn-alpha 2-glycoprotein antiserum. Finally, in vitro translation of prostatic poly(A) + RNA in rabbit reticulocyte lysate in the presence of canine pancreatic microsomal membranes resulted in the formation of an immunoprecipitable 42-kDa band. These results show that Zn-alpha 2-glycoprotein can be synthesized in the prostate itself. The demonstration of high concentrations of this protein in prostatic tissue and prostatic secretion should facilitate the elucidation of its role in the prostate and in other tissues.

Amino Acids↗
PubMed1997

Immunodetection of glycoprotein hormone subunits in nonfunctioning and glycoprotein hormone-secreting pituitary adenomas.

While it is established that nonfunctioning pituitary adenomas (NFPA) produce a spectrum of glycoprotein hormones, the ability of glycoprotein hormone-secreting adenomas to synthesize hormones other than those in vivo hypersecreted has been poorly investigated so far. In this study the immunolocalization of the beta-subunits of LH, FSH, CG and TSH and the common alpha-subunit was investigated in 10 NFPA, 3 gonadotropin-secreting adenomas (Gn-omas, 1 LH-oma, 1 FSH-oma and 1 LH/FSH-oma) and 3 TSH-secreting adenomas (TSH-omas) using an immunohistochemical technique with specific antibodies to glycoprotein subunits. The percentage of positive cells was determined observing at least 5 photograph fields containing about 50 cells. Nine of the 10 NFPA were positive for FSH beta with a high percent of positive cells (> or =30%), 6 for LH beta and 5 for TSH beta with a lower number of labelled cells (from 5 to 30%). Of the 8 NFPA tested, 5 showed a marked positivity for CG beta. In Gn-omas, tumors were positive for the gonadotropin that was in vivo hypersecreted (10-60% positive cells) and negative for either TSH beta or CG beta. Similarly, in TSH-omas TSH beta was detected in a variable proportion of cells (10-30%) with no immunoreactivity for either LH beta or FSH beta or CG beta. This study indicates that the coexpression of FSH beta, LH beta, CG beta and TSH beta molecules is characteristic of NFPA since Gn-omas were negative for CG beta and TSH beta and TSH-omas were negative for FSH beta, LH beta and CG beta. Moreover, the biological events that make NFPA 'silent' glycoprotein hormone-secreting adenomas remain unclear since the positivity for gonadotropins and TSH showed by NFPA was similar to that observed in Gn-omas and TSH-omas, respectively.

Adenoma↗
PubMed1986

Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.

Binding Sites↗
PubMed1984

The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins.

A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non-precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb-IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.

Animals↗