Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes.

Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.     

Demonstration of three major species of pseudorabies virus glycoproteins and identification of a disulfide-linked glycoprotein complex.

The glycoproteins of pseudorabies virus (PRV) Phylaxia were characterized with monoclonal antibodies as specific reagents. Three major structural glycoproteins with molecular weights of 155,000 (155K) (gC), 122K (gA), and 90K (gB) could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We investigated the processing of glycoproteins gA, gB, and gC by in vitro translation, pulse-chase experiments, and in the presence of the ionophore monensin which inhibits glycosylation. gA and gB were found to compose a single polypeptide, whereas gC was found to be a disulfide-linked glycoprotein complex. Immunoprecipitates formed with the aid of anti-gC monoclonal antibodies gave rise to three glycoprotein bands (gC0 [120K], gC1 [67K], and gC2 [58K]) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Limited proteolysis of gC0, gC1, and gC2 resulted in peptide maps of gC0 related to those of both gC1 and gC2. No common peptide bands between gC1 and gC2, however, were seen. We suggest that (i) gC1 and gC2 arise by proteolytic cleavage from the same precursor molecule and stay joined via disulfide bridges and (ii) gC0 is an uncleaved precursor.     

Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gp1 and gp3

Two monoclonal antibodies were prepared against varicella-zoster virus proteins. One of the monoclonal antibodies (10.2) reacted only with the nuclei of infected cells and immunoprecipitated one nonglycosylated late viral protein (125,000 molecular weight). The other monoclonal antibody (19.1) with neutralizing activity, reacted with membrane antigens of infected cells and with the varicella-zoster virus envelope and immunoprecipitated two late major viral glycoproteins (gp1 and gp3). Synthesis of the 125,000-molecular-weight protein, gp1, and gp3 began at 20 to 22 h postinfection, 2 h after the peak of viral DNA synthesis, and continued until 29 h postinfection, when the first progeny virus appeared in infected cells. Pulse-chase experiments showed that during pulse-labeling, only gp1 was detected, whereas during the chase period, gp1 as well as gp3 was detected in infected cells. Under nonreducing conditions, gp3 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 130,000-molecular-weight protein as compared with the 62,000-molecular-weight species obtained when gels were resolved under reducing conditions. This finding indicates that gp3 is a dimer that is disulfide linked.     

Processing of glycoprotein gB related to neutralization of Marek's disease virus and herpesvirus of turkeys

The glycoprotein gB related to neutralization of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) is composed of several glycosylated polypeptides, which were immunoprecipitated with monoclonal antibodies and rabbit antiserum cross-reactive to MDV-gB and HVT-gB, and analyzed by SDS-polyacrylamide gel electrophoresis. The present pulse-chase experiments showed that the precursor forms of MDV- and HVT-gB were glycoproteins with molecular weights of 110K to 115K (gp115/110) and 115K (gp115), respectively. These precursor forms were processed to smaller gB's (gp63 and gp50 for MDV; gp62, gp52, and gp48 for HVT), at least in part by sialylation. The proteins synthesized in the presence of tunicamycin were two polypeptides of 88K and 83K in MDV-infected cells and a 90K polypeptide in HVT-infected cells, indicating the presence of unglycosylated precursor forms of MDV- and HVT-gB. Differences between virulent and avirulent MDV's and between HVT's with and without protective activity against Marek's disease were observed in the processed forms of MDV- and HVT-gB, especially at the processing step of sialylation.     

Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex.

The effect of the carboxylic ionophore monensin on the maturation of Uukuniemi virus, a bunyavirus, and the transport of its two membrane glycoproteins, G1 and G2, were studied in chicken embryo fibroblasts and baby hamster kidney cells. Virus maturation, which occurs in the Golgi complex (E. Kuismanen, K. Hedman, J. Saraste, and R. F. Pettersson, Mol. Cell. Biol. 2:1444-1458, 1982; E. Kuismanen, B. B?ng, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984), was effectively inhibited by the drug (1 or 10 microM) as studied by electron microscopy and by assaying the release of infectious or radiolabeled virus. Immunoelectron microscopy showed that association of viral nucleocapsids with the cytoplasmic surface of glycoprotein-containing Golgi membranes, a prerequisite for virus budding, was unaffected by monensin. In the presence of the drug, the virus glycoproteins assembled into long, tubular structures extending into the lumen of Golgi-derived vacuoles, suggesting that monensin inhibited a terminal step in the assembly of the virus. Intracellular transport and expression of the virus membrane glycoproteins G1 and G2 at the cell surface were not inhibited by monensin as studied by immunocytochemical and radiolabeling techniques. Pulse-chase experiments in the presence of monensin showed that intracellular G1 acquired only partially endo-H-resistant glycans. The sialylation of G1 appearing on the cell surface in the presence of the drug was decreased, whereas sialylation of G2 apparently was inhibited to a lesser extent, as shown by external labeling of the cells with the periodate-boro[3H]hydride method. Thus, monensin exerted a differential effect on the terminal glycosylation of G1 and G2. Unlike several membrane and secretory glycoproteins, both G1 and G2 could enter a functional transport pathway in the presence of monensin and become expressed at the cell surface.     

Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane.

The Rh D blood-group antigen forms part of a complex, involving several other polypeptides, that is deficient in the red cells of individuals who lack all the antigens of the Rh blood-group system (Rhnull red cells). These include components recognized by anti-(Rh D) antibodies and the murine monoclonal antibodies R6A and BRIC 125. We have carried out protein-sequence studies on the components immunoprecipitated by these antibodies. Anti-(Rh D) antibodies immunoprecipitate an Mr-30,000-32,000 polypeptide (the D30 polypeptide) and an Mr-45,000-100,000 glycoprotein (D50 polypeptide). Antibody R6A immunoprecipitates two glycoproteins of Mr 31,000-34,000 (R6A32 polypeptide) and Mr 35,000-52,000 (R6A45 polypeptide). The D30 and R6A32 polypeptides were found to have the same N-terminal amino acid sequences, showing that they are closely related proteins. The D50 polypeptide and the R6A45 polypeptide also had indistinguishable N-terminal amino acid sequences that differed from that of the D30 and R6A32 polypeptides. The putative N-terminal membrane-spanning segments of the two groups of proteins showed homology in their amino acid sequence, which may account for the association of each of the pairs of proteins during co-precipitation by the antibodies. Supplementary data related to the protein sequence have been deposited as Supplementary Publication SUP 50417 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

Characterization of polypeptides immunoprecipitable from Pichinde virus-infected BHK-21 cells

Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.     

Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.     

Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity.     

Association of newly synthesized poly(A) polymerase with four distinct polypeptides

Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.     

The estrogen-responsive 110K and 74K rat uterine secretory proteins are structurally related to complement component C3

Estrogens stimulate the synthesis of specific secretory proteins in the rat uterus. Here we show that two of these, polypeptides of relative molecular weight 110,000 (110K) and 74,000 (74K), are structurally related to C3, the third component of complement, a glycoprotein that plays a central role in regulating complement-mediated inflammatory and immune responses. The similarities were based on the observations that (1) NH2-terminal amino acid sequence of the 74K polypeptide showed sequence homology with the beta chain of mouse C3, (2) comparison of the electrophoretic mobilities of the 110K and 74K polypeptides in the presence and absence of reducing agents revealed that they were disulfide-linked subunits of a protein of Mr approximately 180,000, (3) the native protein was immunoreactive with antibodies specific for rat C3, and (4) both polypeptides were immunoprecipitated with antibodies to rat C3.     

Determinants of Neutralization Resistance in the Envelope Glycoproteins of a Simian-Human Immunodeficiency Virus Passaged In Vivo

In vivo passage of a simian-human immunodeficiency virus (SHIV-89.6) generated a virus, SHIV-89.6P, that exhibited increased resistance to some neutralizing antibodies (G. B. Karlsson et al., J. Exp. Med. 188:1159-1171, 1998). Here we examine the range of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies to which the passaged virus became resistant and identify envelope glycoprotein determinants of antibody resistance. Compared with the envelope glycoproteins derived from the parental SHIV-89.6, the envelope glycoproteins of the passaged virus were resistant to antibodies directed against the gp120 V3 variable loop and the CD4 binding site. By contrast, both viral envelope glycoproteins were equally sensitive to neutralization by two antibodies, 2G12 and 2F5, that recognize poorly immunogenic structures on gp120 and gp41, respectively. Changes in the V2 and V3 variable loops of gp120 were necessary and sufficient for full resistance to the IgG1b12 antibody, which is directed against the CD4 binding site. Changes in the V3 loop specified complete resistance to a V3 loop-directed antibody, while changes in the V1/V2 loops conferred partial resistance to this antibody. The epitopes of the neutralizing antibodies were not disrupted by the resistance-associated changes. These results indicate that in vivo selection occurs for HIV-1 envelope glycoproteins with variable loop conformations that restrict the access of antibodies to immunogenic neutralization epitopes.     

Limited proteolysis. Early steps in the processing of large premature termination fragments of beta-galactosidase in Escherichia coli

The mechanism by which large premature termination fragments of beta-galactosidase were degraded in Escherichia coli was studied using quantitative immunoprecipitation techniques. Two different lacZ nonsense mutants which produced apparent primary translation products of 96,000 and 109,000 daltons, respectively, were both shown to produce a second beta-galactosidase-related polypeptide of Mr = 90,000. These 90,000-dalton polypeptides appeared to be the same in both strains since they co-migrated when analyzed as a mixture on sodium dodecyl sulfate-polyacrylamide gels and were indistinguishable when analyzed by one-dimensional peptide mapping. Pulse-chase experiments established a stoichiometric precursor-product relationship between the primary mutant gene products (called the A polypeptides) and the common 90,000-dalton polypeptide (called the B polypeptide). No intermediates were detected between the A and B polypeptides. We propose that there is a common pathway for the degradation of these different large fragments of beta-galactosidase. According to this model, the first step would be a specific endoproteolytic cleavage of the primary translation product which produces the 90,000-dalton polypeptide as a common intermediate. The kinetic analysis demonstrated a first order decay of both A and B polypeptides but, surprisingly, the first order rate constant for the decay of A appeared dependent upon the induction regimen. This result suggested that degradation may possibly be autoregulated either by the intracellular level of A or by other intermediates in the degradation pathway.     

Separation of neutralizing and hemagglutination-inhibiting antibody activities and specificity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions.

Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived polyoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying 125I-labeled immunoglobulins to SDS-polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacrylamide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutralizing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immunoprecipitation experiments to demonstrate that the antigenic determinants responsible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both of these polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1.     

牛病毒性腹泻病毒单克隆抗体的研究

牛病毒性腹泻——粘膜病是世界性广泛流行的奶牛和肉牛的传染病。其病原为牛病毒性腹泻病毒(BVDV),属于披膜病毒科的瘟病毒属,它的许多生物学特性至今还不很清楚。本试验建立了12株分泌抗BVDV的单克隆抗体(McAb)杂交瘤细胞株,并结合免疫转移电泳法和放射免疫沉淀法,初步研究了BVDV的多肽。     

Schistosoma mansoni: reactivity with infected human sera and monoclonal antibody characterization of a glycoprotein in different developmental stages

Cercarial glycoproteins of Schistosoma mansoni were purified by concanavalin A affinity chromatography. The purified fraction consisted of at least 15 polypeptides when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of infected humans specifically immunoprecipitated all of these polypeptides. These purified glycoproteins were used as antigen for preparing monoclonal antibodies. One of these monoclonal antibodies immunoprecipitated cercarial polypeptides that were identical to polypeptides immunoprecipitated with sera of infected humans as analyzed by two-dimensional gel electrophoresis. Direct binding assays with ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present not only in cercariae (the source of the immunogen) but also in adult male and female worms and in eggs. The protein molecules expressing these antigenic determinants were glycosylated in each of the developmental stages of the larvae, but differed with respect to molecular weight. These findings indicate a role for this monoclonal antibody in serodiagnosis and immunoprophylaxis.     

Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.