Synthesis, cellular location, and immunogenicity of bovine herpesvirus 1 glycoproteins gI and gIII expressed by recombinant vaccinia virus.

Two of the major glycoproteins of bovine herpesvirus 1 (BHV-1) are gI, a polypeptide complex with apparent molecular weights of 130,000, 74,000, and 55,000, and gIII (a 91,000-molecular-weight [91K] glycoprotein), which also exists as a 180K dimer. Vaccinia virus (VAC) recombinants were constructed which carry full-length gI (VAC-I) or gIII (VAC-III) genes. The genes for gI and gIII were each placed under the control of the early VAC 7.5K gene promoter and inserted within the VAC gene for thymidine kinase. The recombinant viruses VAC-I and VAC-III retained infectivity and expressed both precursor and mature forms of glycoproteins gI and gIII. The polypeptide backbones, partially glycosylated precursors, and mature gI and gIII glycoproteins were indistinguishable from those produced in BHV-1-infected cells. Consequently, they were apparently cleaved, glycosylated, and transported in a manner similar to that seen during authentic BHV-1 infection, although the processing efficiencies of both gI and gIII were generally higher in recombinant-infected cells than in BHV-1-infected cells. Immunofluorescence studies further demonstrated that the mature gI and gIII glycoproteins were transported to and expressed on the surface of cells infected with the respective recombinants. Immunization of cattle with recombinant viruses VAC-I and VAC-III resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gI and gIII. These data demonstrate the immunogenicity of VAC-expressed gI and gIII and indicate the potential of these recombinant glycoproteins as a vaccine against BHV-1.     

Estrogen induces N-linked glycoprotein expression by immature mouse uterine epithelial cells

Characterization of complex glycoconjugates and the effects of estrogen on their expression in immature mouse uterine epithelial cells are reported. The secreted fraction contained nonanionic, O-linked lactosaminoglycan (LAG)-bearing proteins of Mr 30,000-40,000 as well as anionic, O-linked, LAG-bearing glycoproteins with very high apparent molecular weight (greater than 670K). Heparan sulfate (HS) proteoglycans and HS linked to little or no protein were found in the secreted fraction as well. A very similar array of glycoconjugates was found in the nonhydrophobic fraction of cell-associated macromolecules. In addition, the hydrophobic cell-associated fraction contained nonanionic, LAG-bearing glycoproteins of approximately 250K, anionic LAG-bearing glycoproteins distributing over a wide range of molecular weights, and HS proteoglycans with median molecular weights of approximately 250K. In contrast to the glycoproteins produced by their mature counterparts, virtually all glycoproteins produced by immature cells were O-linked. Estrogen treatment of immature mice caused uterine epithelial cells to secrete anionic, high molecular weight (greater than 670K) N-linked glycoproteins as a major product. These estrogen-responsive glycoproteins did not appear to contain LAGs. Estrogen treatment also markedly decreased the proportion of all hydrophobic glycoconjugates in the cell-associated fraction. Collectively, these observations indicate that one aspect of the estrogen-induced maturation of uterine epithelial cells is the stimulation of N-linked glycoprotein synthesis and secretion. Furthermore, stimulation of N-linked glycoprotein synthesis by itself is insufficient to support N-linked LAG glycoprotein production.     

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.     

Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.     

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.     

Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods.

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.     

Sequence and expression of a bovine herpesvirus-1 gene homologous to the glycoprotein K-encoding gene of herpes simplex virus-1

In the bovine herpesvirus-1 (BHV-1) genome, a gene equivalent to the glycoprotein K (gK)-encoding gene of other herpesviruses was identified and sequenced. The primary translation product is predicted to comprise 338 amino acids (aa) and to exhibit a molecular mass of 37.5 kDa. It possesses characteristics typical for membrane glycoproteins including a potential cleavable signal sequence, three transmembrane domains and two potential N-linked glycosylation sites. Comparison to the gK proteins of other herpesviruses revealed aa sequence homologies of 46, 44, 53, 43 and 46% with the gK counterparts of herpes simplex viruses-1 and 2 (HSV-1 and 2), equine herpesvirus-1 (EHV-1), Marek's disease virus (MDV) and varicella zoster virus (VZV), respectively. A 30-kDa primary translation product was identified following in vitro translation of in vitro transcribed mRNA. When canine microsomal membranes were added to the translation reaction, a 38-kDa glycosylated protein was detected. Treatment with endoglycosidase For H (endo For H) removed the glycosyl groups and reduced the apparent molecular mass of the 38-kDa glycoprotein.     

Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.     

On the macromolecular composition of the zona pellucida from porcine oocytes

The chemical and immunological relation between the glycoprotein components of the pig oocyte zona pellucida resolved by two-dimensional polyacrylamide gel electrophoresis was investigated. After disulfide bond reduction, four microheterogeneous glycoprotein components with apparent molecular weights of 25K, 55K, 65K, and 90K were resolved. When disulfide bonds were left unreduced, two microheterogeneous glycoprotein components were resolved with apparent molecular weights of 55K and 90K. Actin was present, but as a contaminant of the zona pellucida rather than as a true component. The structural relation of these components was investigated using deglycosylation with trifluoromethane-sulfonic acid, limited proteolysis with Staphylococcus aureus V8 protease, amino acid and carbohydrate composition analyses, sequence analysis, and monoclonal antibodies. The 25K and 65K components comigrated with the 90K component when disulfide bonds were not reduced. When the intermolecular disulfide bonds crosslinking the two components were reduced, the 25K and 65K components behaved independently. The 25K and 65K components were derived from the 90K glycoprotein family by proteolysis. The 25K component originated from the C-terminal end, and the 65K component from the N-terminal end of the 90K glycoprotein. The 55K component was composed of two chemically and antigenically distinct glycoproteins, termed 55K alpha and 55K beta, that electrophoretically comigrated. The N-terminal amino acid of the 55K alpha family was blocked. The 55K beta family had an N-terminal amino acid sequence of Asp-Val-Pro-Thr-Ile-Gly-Leu-Ser-X-Ala-Pro-Thr. Thus, the two to four electrophoretic components of the zona pellucida observed on gel electrophoresis are derived from three glycoprotein families.     

Structural glycoproteins from rabbit aortic media.

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.     

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.     

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.     

High molecular weight forms of adrenocorticotropic hormone are glycoproteins.

Mouse pituitary tumor cells (AtT-20/D-16v) were incubated in medium containing [3H] glucosamine or [3H] mannose. By analyzing immunoprecipitates of cell extracts and culture medium it was shown that [3H] glucosamine and [3H] mannose were incorporated into all three high molecular weight forms of ACTH; label was not incorporated into Mr=4,500 ACTH (which is thought to be similar to the 39 amino acid polypeptide form of ACTH, alpha(1-39)). Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apparent molecular weights of these glycoprotein forms of ACTH were 31,000, 23,000, and 13,000. Gel filtration in 6 M guanidine HCl indicated that the molecular weights of these forms of ACTH were substantially lower; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has often been found to overestimate the molecular weight of glycoproteins. A significant fraction of the high molecular weight ACTH in tumor cell extracts binds to columns of concanavalin A-agarose and can be eluted with 0.2 M alpha-methyl-D-mannopyranoside; porcine alpha(1-39) does not bind to concanavalin A-agarose. High molecular weight glycoprotein ACTH can be detected in extracts of mouse and bovine pituitary by using concavalin A affinity chromatography.     

Purification and characterization of 31-kDa palm pollen glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients

A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4% yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the beta1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having alpha1-3 fucose and alpha1-2 xylose residues (GlcNAc(2 to approximately 0)Man3Xyl1Fuc(1 to approximately 0)GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man(9 to approximately 5)GlcNAc2).     

Platelet plasma membrane glycoproteins Identification of a proteolytic substrate for thrombin

The surface glycoproteins of the platelet plasma membrane were labeled by oxidation with galactose oxidase followed by reduction with (³H)-sodium borohydride. Of the glycoproteins labeled, only glycoprotein V (apparent molecular weight of 89,000) was decreased as a result of thrombin action. The affected glycoprotein appeared to be completely removed at a concentration of 1 U thrombin per 10⁹ platelets. A soluble glycopeptide hydrolytic product with an apparent molecular weight of 70,000 was released into solution.     

Biochemical studies on the Phlebotomus fever group viruses (Bunyaviridae family).

Analyses of the virion polypeptides and genomes of several Phlebotomus fever group viruses, Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses, have established that they are biochemically similar to the accepted members of the Bunyaviridae family. Like snowshoe hare virus (a member of the California serogroup of the Bunyavirus genus of the Bunyaviridae family), Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses all have three viral RNA species, designated large (L), medium (M), and small (S). Oligonucleotide fingerprint analyses of Karimabad and Punta Toro virus RNA species indicated that their L, M, and S RNA species are unique. By polyacrylamide gel electrophoresis it was determined for Karimabad virus that the apparent molecular weights of its L, M, and S RNA species are 2.6 X 10(6), 2.2 X 10(6), and 0.8 X 10(6), respectively. For Punta Toro virus, the apparent molecular weights of its L, M, and S RNA species are 2.8 X 10(6), 1.8 X 10(6), and 0.75 X 10(6), respectively. The major internal nucleocapsid (N) protein of Karimabad virus was found to have a molecular weight of 21 X 10(3). A similar polypeptide size class was identified in preparations of sandfly fever Sicilian serotype, Chagres, and Punta Toro viruses. The Karimabad virus glycoproteins formed the external surface projections on virus particles and could be removed from virus preparations by protease treatment. The glycoproteins in an unreduced sample could be resolved into two size classes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They had apparent molecular weights of 62 X 10(3) and 50 X 10(3) in continuous polyacrylamide gels. When Karimabad virus preparations were reduced with 1% beta-mercaptoethanol, prior to resolution by continuous polyacrylamide gel electrophoresis, all the viral glycoprotein was recovered in a single size class, having an apparent molecular weight of 62 X 10(3). Two or three major virion polypeptides have been identified in preparations of Punta Toro, Chagres, and sandfly fever Sicilian serotype viruses.     

Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.     

Lens glycoproteins: biosynthesis in cultured epithelial cells of bovine lens.

1. Radioactivity from [3H]glucosamine is rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated isotope appears largely in glycoproteins of the cell surface that are exposed to trypsin and are released into a soluble form by proteolysis of intact cells. Glycoproteins are also secreted by cultured cells and can be recovered in the culture fluids. Sodium dodecysulphate-polyacrylamide gell electrophoresis shows that a range of glycoproteins with apparent molecular weights from approximately 14000 to 120000 are present. The relationships of these glycoproteins to collagen and the non-collagenous glycoproteins of lens basement membranes are discussed. 2. A plasma membrane fraction obtained from non-trypsinised lens epithelial cells contains one major glycoprotein of apparent molecular weight 120000. A major non-glycosylated polypeptide of molecular weight about 38000 detectable by Bloemendal et al. (1972) in plasma membranes of differentiated lens fibre cells was not prominent in lens epithelial cell membranes. 3. Examination of lens basement membranes extracted in various ways failed to reveal major glycoproteins of low molecular weight. Higher molecular weight glycoproteins, some of them related to collagen, were present.     

Analysis of Human T-Cell Antigens by One- and Two-Dimesional Polyacrylamide Gel Electrophoresis

One- and two-dimensional polyacrylamide gel electrophoresis (PAGE) was performed on immunoprecipitates formed between anti-human T-cell xenoantiserum (ATS) and cell-surface glycoproteins of human lymphocytes, that had been radioiodinated by lactoperoxidase and purified on a Lentil lectin-coupled Sepharose 4B column. In some experiments, the cells were ³H-labeled by periodate-tritiated borohydride. ATS that was absorbed with B cells recognized a number of cell-surface antigens expressed preferentially on human thymus and T cells, with molecular weights of 150K (T150), 94K (T94), 72K (T72), and 65K (T65) daltons. Whereas T150 appeared to consist of multiple components of heavily sialylated glycoproteins and to be expressed largely on thymus and T cells, and to a much lesser extent on B cells, the remaining T94, T72, and T65 glycoproteins seemed to be present on thymus and T cells but absent from B cells. Two-dimentional PAGE analysis of these T-cell glycoproteins precipitated by ATS demonstrated that T94 was an acidic glycoprotein with pI of 4, while T72 and T65, the latter being found on thymus and T cells but not on T cell-type leukemic cells, exhibited marked electric charge heterogeneity with pI ranging from 4 to 7. These data clearly suggest that human thymus and T cells possess a complex antigenic make-up on their cell surfaces, comparable to that of mouse T cells with a variety of Ly antigen systems.