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PubMed1990

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion.

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.

Amino Acid Sequence↗
PubMed1987

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment.

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.

Acetylglucosaminidase↗
PubMed1990

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.

Adult↗
PubMed1996

Vaccination with a cocktail of seven recombinantly expressed HSV-1 glycoproteins protects against ocular HSV-1 challenge more efficiently than vaccination with any individual glycoprotein.

Our previous studies have shown that of seven HSV-1 glycoproteins (gB, gC, gD, gE, gG, gH and gI) individually expressed in baculovirus, vaccination with gD provides the best protection against HSV-1 challenge. To establish whether vaccination with a mixture of these seven expressed glycoproteins would provide better protection against HSV-1 challenge than vaccination with gD alone, we determined the level of protection afforded by vaccination with a cocktail of the seven expressed glycoproteins. The amount of each of the seven expressed glycoproteins in the mixture was equivalent to one-seventh the amount of gD used in the gD alone vaccination. Thus, the total amount of glycoprotein was the same for the cocktail and gD alone vaccine. For neutralizing antibody titer, delayed-type hypersensitivity (DTH), and survival following lethal challenge, no difference was observed between mice vaccinated with all seven glycoproteins and those vaccinated with gD. However, for other criteria, vaccination with all seven glycoproteins appeared to provide better protection than vaccination with gD. Following ocular challenge, virus was not detected at any time in the tears of mice vaccinated with all seven glycoproteins. In contrast, virus was detected in the tears of gD vaccinated mice for up to 3 days post challenge. Mock vaccinated mice had virus in their tears for as long as 10 days. Mice vaccinated with all seven glycoproteins had no eye disease, while gD vaccinated mice had a significant amount of blepharitis. Finally, compared to gD vaccinated mice, the mice vaccinated with all seven glycoproteins were more efficiently protected against the establishment of HSV-1 latency following ocular infection. Our results therefore suggest that while for some protective criteria there was no significant difference between vaccination with gD or seven glycoproteins, vaccination with seven glycoproteins was more efficient in protecting challenged mice against some forms of eye disease, the duration of infection and the establishment of latency.

Animals↗
PubMed1995

Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.

Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.

Amino Acid Sequence↗
PubMed1987

The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells.

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.

1-Deoxynojirimycin↗
PubMed2001

Evidence that natural killer cells express mini P-glycoproteins but not classic 170 kDa P-glycoprotein.

Several lines of evidence including reverse transcription polymerase chain reaction, immunoreactivity and their ability to efflux rhodamine 123 have implied the existence of P-glycoprotein in natural killer (NK) cells. It has been a natural tendency to assume that NK-cell P-glycoprotein is identical to the P-glycoprotein of multidrug resistant (MDR) cell lines, however, the present study uncovered major differences. Functionally, NK cells demonstrated a restricted substrate profile, being unable to transport daunorubicin and calcein acetoxymethylester while efficiently transporting other P-glycoprotein substrates. Furthermore, physical differences in NK-cell P-glycoprotein were established by differential reactivity with P-glycoprotein antibodies. NK cells demonstrated strong reactivity with C494 and JSB-1, but did not react appreciably with C219. In addition, NK cells were unable to bind to the antibody MM4.17 unless they had been fixed and permeabilized, yet this antibody normally recognizes an extracellular epitope of P-glycoprotein. These differences culminated in the demonstration using Western analysis that NK cells did not express detectable levels of 170 kDa P-glycoprotein. Instead, NK cells expressed small-molecular-weight 'mini P-glycoprotein' products, of approximately 70 and 80 kDa. Collectively, these data indicate that the predominant P-glycoprotein species of NK cells are novel mini P-glycoproteins and not the classic P-glycoprotein of MDR models.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1995

Synchronized inhibition of the phospholipid mediated autoactivation of factor XII in plasma by beta 2-glycoprotein I and anti-beta 2-glycoprotein I.

Lupus anticoagulants are a group of antibodies commonly found in patients with autoimmune diseases such as systemic lupus erythematosus. Lupus anticoagulants inhibit phospholipid dependent coagulation and may bind to negatively charged phospholipids. Recent studies have suggested an association between anti-beta 2-glycoprotein I and a lupus anticoagulant, whose activity is frequently dependent on the presence of beta 2-glycoprotein I. Based on these observations, the effect of anti-beta 2-glycoprotein I on the autoactivation of factor XII in plasma was investigated. Autoactivation initiated by the presence of negatively charged phospholipids, but not by sulfatide, was strongly inhibited by immunoaffinity purified anti-beta 2-glycoprotein I. The dose-response curve of anti-beta 2-glycoprotein I was identical with that of a precipitating antibody, showing no inhibition at low and high antibody dilutions and maximal inhibition at an intermediate dilution. At high antibody concentrations, an increased rate of factor XIIa activation was observed. This increase was of the same magnitude as the decreased rate observed in plasma supplemented with the same amount of beta 2-glycoprotein I as in the plasma itself. This confirms the inhibitory effect of beta 2-GP-I on the contact activation and shows that inhibition is effective on the autoactivation of factor XII in plasma. The inhibitory action of beta 2-glycoprotein I was independent of the inhibition caused by the anti-beta 2-glycoprotein I/beta 2 glycoprotein I complex suggesting a synchronized inhibition of factor XII autoactivation by beta 2-glycoprotein I and anti-beta 2-glycoprotein I. The inhibition caused by the antibody is suggested to be caused by a reduced availability of negatively charged phospholipids due to the binding of the anti-beta 2-GP-I/beta 2-GP-I complex. This complex may be a lupus anticoagulant.

Animals↗
PubMed1989

Platelet membrane glycoprotein V: characterization of the thrombin-sensitive glycoprotein from human platelets.

Human platelets contain a single membrane glycoprotein which is susceptible to thrombin proteolysis, glycoprotein V. We have purified 1 mg of glycoprotein V from 10(13) platelets using a combination of gel filtration, hydroxylapatite and ion-exchange chromatographies. Glycoprotein V has a blocked amino-terminus. Following proteolysis by human alpha-thrombin, a major fragment, termed glycoprotein Vf1, had the sequence Gly-Pro-Phe-X-Arg-Pro-Ala-Ala-Asp-Glu-Ser-Val-Glu-Ala-Pro-Val-Asn-Gln-Al a-Glu- Ala-Pro-. The purified glycoprotein was not a substrate for human gamma-thrombin. Glycoprotein V contained 17.5% carbohydrate, with the majority of the carbohydrate consisting of neutral hexoses. Deglycosylated glycoprotein V had a molecular weight of 57.5 kDa compared to the glycosylated protein's 82 kDa and the deglycosylated protein was recognized by polyclonal antibodies raised against glycoprotein V. Immunoelectrophoresis of human and rat platelets and megakaryocytes gave a single immunoreactive band, with the rat glycoprotein having a slightly larger molecular mass. Glycoprotein V is most likely an integral membrane protein.

Amino Acid Sequence↗
PubMed1986

Purification of individual varicella-zoster virus (VZV) glycoproteins gpI, gpII, and gpIII and their use in ELISA for detection of VZV glycoprotein-specific antibodies.

We have utilized monoclonal antibodies in immune affinity chromatography to purify each of the 3 major glycoproteins of varicella-zoster virus (VZV), gpI, gpII, and gpIII, in immunologically active form. Upon injection into guinea pigs, each preparation elicited the production of specific antibodies capable of immunoprecipitating the homologous glycoprotein and of neutralizing VZV infectivity in vitro. Also, total glycoproteins from VZV-infected cells have been purified by lectin affinity chromatography. Each of the individual purified glycoproteins, as well as total VZV glycoproteins and appropriate uninfected cell protein controls, have been employed as solid-phase reagents in enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies directed against specific VZV glycoproteins. The specificity of the purified glycoproteins as ELISA reagents was verified by the ability of individual monoclonal antibodies to bind specifically to individual glycoprotein preparations. We have demonstrated the utility of the glycoprotein-specific ELISA by detecting antibodies in sera from post-zoster and post-varicella patients. The assay detects antibodies directed against each of the 3 major glycoproteins and is sensitive enough to detect antibodies in a 1:320 000 dilution of some sera. This assay, as well as the purified individual glycoproteins per se, should prove to be very useful reagents in understanding the role of each of gpI, gpII, and gpIII in immunity to VZV.

Animals↗
PubMed1998

beta2-glycoprotein-I (apolipoprotein H) and beta2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor megalin.

Screening of serum by using a surface plasmon resonance analysis assay identified beta2-glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated beta2-glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of beta2-glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for beta2-glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when beta2-glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of beta2-glycoprotein-I and beta2-glycoprotein-I- phospholipid complexes to megalin was completely blocked by receptor-associated protein. In conclusion, we have demonstrated a novel receptor recognition feature of beta2-glycoprotein-I. In addition to explaining the high urinary excretion of beta2-glycoprotein-I in patients with renal tubule failure, the data provide molecular evidence for the suggested function of beta2-glycoprotein-I as a linking molecule mediating cellular recognition of phosphatidylserine-exposing particles.

Animals↗
PubMed2002

P-glycoprotein-actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin.

P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump-related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1976

Isolation and characterization of an epithelial basement membrane glycoprotein from murine kidney and further characterization of an epithelial basement membrane glycoprotein secreted by murine teratocarcinoma cells in vitro.

A glycoprotein component of epithelial basement membranes (EBM) has been isolated from murine kidney homogenates by extraction with 0.05 M phosphate buffer, pH 7.2, precipitation with (NH4)2SO4 and chromatography on controlled pore glass. Antiserum produced against this glycoprotein reacts specifically with the basement membranes of renal glomeruli and tublules. The EBM glycoprotein of renal origin is antigenically identical with a glycoprotein component of epithelial basement membrane secreted by a murine teratocarcinoma grown in vitro, and the amino acid composition of the two EBM glycoproteins is markedly similar. Both glycoproteins were isolated as high molecular weight aggregates. Disaggregation with sodium dodecyl sulfate and 2-mercaptoethanol resulted in release of monomers of 32 000 and 34 000 daltons for kidney EBM glycoprotein and teratocarcinoma EBM glycoprotein, respectively. The difference in molecular weight is apparently due to increased amounts of fucose, mannose, N-acetylglucosamine and sialic acid in the glycoprotein secreted by the teratocarcinoma. In addition, both EBM glycoproteins contain galactose, glucose and N-acetylgalactosamine.

Amino Acids↗
PubMed1978

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins.

Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.

Animals↗
PubMed1975

Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins.

We have reported the isolation of a clone (termed 15B) of Chinese hamster ovary (CHO) cells which are deficient in certain plant lectin-binding sites and have decreased amounts of sialic acid, galactose, and N-acetylglucosamine in its membranes (Gottlieb et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1078-1082). This study demonstrates that extracts of 15B cells, in contrast to the parent cell line, do not transfer N-acetylglucosamine residues from UDP-GlcNAc to certain glycopeptide and glycoprotein acceptors containing terminal nonreducing alpha-linked mannose residues. The decreased enzyme activity could not be accounted for by the presence of inhibitors, altered pH, or Mn2+ requirements of the glycosyltransferase or increased N-acetylglucosaminidase activity in the extracts. The finding that the 15B cell extracts have significant but reduced N-acetylglucosaminyltransferase activity toward a degraded orosomucoid acceptor suggests that these cells have a selective loss of one of several specific N-acetylglucosaminyltransferases which are present in the parent CHO cells. The sialyl- and galactosyltransferase activities of 15B and parent CHO cells are comparable. Parent CHO and 15B cells were grown in radioactive glucosamine to label the membrane glycoproteins. Solubilization of these glycoproteins and passage over a Ricinus communis agglutinin I (RCA I) Sepharose affinity column revealed that no labeled 15B glycoprotein material bound, whereas 50 percent of the CHO membrane glycoproteins bound and could be eluted with the haptene lactose, demonstrating that 15B cells are virtually devoid of membrane oligosacharides capable of binding to the RCA I lectin. The 15B membrane glycoproteins exhibited a marked shift toward glycoprotein species of lower molecular weight when examined by gel electrophoresis in sodium dodecyl sulfate. It is proposed that this shift in the mobility of the 15B membrane glycoproteins results from a decreased glycosylation of a number of membrane glycoproteins relative to their counterparts in CHO cells. The deficient N-acetylglucosaminyltransferase activity in 15B cells can account for the decreased glycosylation of the 15B cell membrane glycoproteins.

Cell Membrane↗
PubMed1991

Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies.

We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells. With low frequency we obtained stable, MDR3-expressing clones. Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated. Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance. The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D. We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy. Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones. Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs. We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting. Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein. In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Characterization of the ATPase activity of the Mr 170,000 to 180,000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells.

The Mr 170,000 to 180,000 membrane glycoprotein associated with multidrug resistance (P-glycoprotein) is involved in drug transport mechanisms across the plasma membrane of multidrug-resistant cells. We have recently reported the purification of P-glycoprotein. The purified P-glycoprotein was found to have an ATPase activity, which might be coupled with the active efflux of anticancer drugs. In the present study, we have further studied the properties of the P-glycoprotein ATPase activity by an immobilized enzyme assay procedure using a P-glycoprotein-antibody-Protein A-Sepharose complex. GTP was also hydrolyzed by the P-glycoprotein, although less efficiently than ATP. The ATPase activity of P-glycoprotein had an optimal pH range around neutrality (pH 6.5-7.4). The detergent concentration of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate used for protein solubilization was essential for enzyme recovery. Maximum activity was obtained when 0.1-0.2% 3-[(3-cholamidopropyl)dimethyl-ammonio]-propane sulfonate was used, while higher concentrations markedly inhibited the ATPase activity. The ATPase activity was dependent on Mg2+; maximum activity was obtained at 2-10 mM. Manganese and cobalt could substitute for magnesium as ionic cofactors. Divalent cations such as Ca2+, Zn2+, Ni2+, Cd2+, and Cu2+ inhibited the Mg2+-catalyzed ATP hydrolysis. N-Ethylmaleimide and vanadate inhibited the ATPase activity, while sodium azide or ouabain had no effect. Anticancer agents such as vincristine and Adriamycin did not affect the enzyme activity. In contrast, verapamil and trifluoperazine, agents which inhibit active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the P-glycoprotein ATPase activity suggesting that P-glycoprotein might be the target molecule of these agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗