Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily.

gp49 is a Mr 49,000 glycoprotein expressed on the surface of mouse bone marrow-derived mast cells, which are progenitors for the major in vivo mast cell subclasses, typified by intestinal mucosal mast cells and serosal mast cells. The amino-terminal amino acid sequence of gp49 was determined after isolation of the solubilized membrane protein by affinity chromatography with the B23.1 anti-gp49 monoclonal antibody. Redundant oligonucleotides were used to isolate a full-length 1.3-kilobase cDNA from a mouse mast cell library. The predicted amino acid sequence contains a signal peptide of 23 residues, an extracellular domain of 215 residues with three potential sites of N-linked glycosylation, a transmembrane domain of 23 residues, and a cytoplasmic tail of 42 residues. Hybridization of the gp49 cDNA was limited to mRNA extracted from those cell types that also bound the B23.1 monoclonal antibody as assessed by cytofluorographic analyses. The predicted extracellular domain of gp49 contains two regions of 48 and 51 amino acids, each flanked by cysteine residues. Both regions meet criteria for being C2-type domains of the immunoglobulin superfamily based upon the alignment of consensus amino acids and their predicted secondary structure organization. Thus, gp49, a membrane glycoprotein preferentially expressed by the progenitor mast cell population, is a new member of the immunoglobulin superfamily.     

Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants.

In this paper we report the nucleotide sequence and predicted amino acid sequence of glycoprotein B of herpes simplex virus 1 strain F and the amino acid substitutions in the domains of the glycoprotein B gene of three mutants selected for resistance to monoclonal antibody H126-5 or H233 but not to both. Analyses of the amino acid sequence with respect to hydropathicity and secondary structure yielded a two-dimensional model of the protein. The model predicts an N-terminal, 29-amino-acid cleavable signal sequence, a 696-amino-acid hydrophilic surface domain containing six potential sites for N-linked glycosylation, a 69-amino-acid hydrophobic domain containing three segments traversing the membrane, and a charged 109-amino-acid domain projecting into the cytoplasm and previously shown to marker rescue glycoprotein B syn mutations. The nucleotide sequence of the mutant glycoprotein B DNA fragments previously shown to marker transfer or rescue the mutations revealed that the amino acid substitutions cluster in the hydrophilic surface domain between amino acids 273 and 305. Analyses of the secondary structure of these regions, coupled with the experimentally derived observation that the H126-5- and H233-antibody cognitive sites do not overlap, indicate the approximate locations of the epitopes of these neutralizing, surface-reacting, and immune-precipitating monoclonal antibodies. The predicted perturbations in the secondary structure introduced by the amino acid substitutions correlate with the extent of loss of reactivity with monoclonal antibodies in various immunoassays.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

Glycoprotein B of herpes simplex virus type 1 oligomerizes through the intermolecular interaction of a 28-amino-acid domain.

Herpes simplex virus type 1 glycoprotein B (gB) is an envelope component that plays an essential role in virus infection. The biologically active form of gB is an oligomer that contributes to the process of viral envelope fusion with the cell surface membrane, resulting in viral penetration and initiation of the replication cycle. In previous studies, two discontinuous sites for oligomer formation were identified: a nonessential upstream site located between residues 93 and 282 and an essential downstream site located between residues 596 and 711. In this study, in vitro-transcribed and -translated gB test molecules were used to characterize the more active essential membrane-proximal domain. A series of gB test polypeptides mutated in this downstream oligomerization domain were assayed for their abilities to form oligomers with a mutant gB capture polypeptide containing the analogous wild-type domain. Detection of oligomers was achieved by coimmunoprecipitation of two gB mutant molecules by using a monoclonal antibody specific for a hemagglutinin epitope tag introduced into the coding sequence of the capture polypeptide. Analysis of the immune-precipitated products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the downstream oligomerization domain resided within residues 626 to 676. This region was further resolved into two segments, residues 626 to 653 and 653 to 675, each of which was independently sufficient to form oligomers. However, residues 626 to 653 provided for a stronger interaction between gB monomers. Moreover, this stretch of 28 amino acids was shown to form oligomers when introduced into the carboxy-terminal region of gB monomers lacking this domain at the normal site, thus indicating that this domain was functionally independent of its natural location within the gB molecule. Further analysis of the sequence within residues 596 to 653 by using mutant test polypeptides altered in individual amino acids revealed that cysteines 9 and 10 located at positions 596 and 633, respectively, were not required for oligomer formation but contributed to dimer formation and/or stabilization. The results of this study suggest that oligomerization of gB monomers is induced by interactions between contiguous residues localized within the ectodomain near the site of molecule insertion into the viral envelope membrane.     

The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria

The hexagonally patterned surface layer of halobacteria consists of a true glycoprotein. This procaryotic glycoprotein has recently been shown to exhibit novel features with respect to saccharide structure and saccharide biosynthesis. The primary structure and the location of glycosylation sites were determined by cloning and sequencing of the glycoprotein gene of Halobacterium halobium. According to the predicted amino acid sequence, the glycoprotein is synthesized with a N-terminal leader sequence of 34 amino acid residues reminiscent of eucaryotic and procaryotic signal peptides. A hydrophobic stretch of 21 amino acid residues at the C terminus probably serves as a transmembrane domain. 14 threonine residues are clustered adjacent to this membrane anchor and linked to these threonines are all the disaccharides of the cell surface glycoprotein. 12 N-glycosylation sites are distributed over the polypeptide chain.     

Characterization of diverse primary herpes simplex virus type 1 gB-specific cytotoxic T-cell response showing a preferential V beta bias.

The glycoprotein B (gB) from herpes simplex virus type I is a major target of cytotoxic T lymphocytes (CTL) in C57BL/6 mice. The majority of these T cells are directed to a single Kb-restricted determinant, gB498-505. We have analyzed the T-cell receptor (TCR) usage in gB-specific CTL lines derived shortly after virus infection. The CTL populations preferentially used two V beta regions, a dominant V beta 10 element and a subdominant V beta 8 element. Detailed sequence analysis revealed considerable TCR beta-chain heterogeneity despite a striking level of predicted amino acid conservation at the V beta-D beta junction. This junction forms part of the third hypervariable loop of the TCR thought to directly contact the major histocompatibility complex-bound antigenic peptide. The results reveal considerable diversity within the primary T cells responding to a single viral determinant while still maintaining a high degree of TCR V beta bias and sequence conservation at the V-D-J junction.     

Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.     

Sequence characteristics of a gene in equine herpesvirus 1 homologous to glycoprotein H of herpes simplex virus.

A gene in equine herpesvirus 1 (EHV-1, equine abortion virus) homologous to the glycoprotein H gene of herpes simplex virus (HSV) was identified and characterised by its nucleotide and derived amino acid sequence. The EHV-1 gH gene is located at 0.47-0.49 map units and contains an open reading frame capable of specifying a polypeptide of 848 amino acids, including N- and C-terminal hydrophobic domains consistent with signal and membrane anchor regions respectively, and 11 potential sites for N-glycosylation. Alignment of the amino acid sequence with those published for HSV gH, varicella zoster virus gpIII, Epstein Barr virus gp85 and human cytomegalovirus p86 shows similarity of the EHV gene with the 2 other alpha-herpesviruses over most of the polypeptide, but only the C-terminal half could be aligned for all 5 viruses. The identical positioning of 6 cysteine residues and a number of highly conserved amino acid motifs supports a common evolutionary origin of this gene and is consistent with its role as an essential glycoprotein of the herpesvirus family. An origin of replication is predicted to occur at approximately 300 nucleotides downstream of the EHV-1 gH coding region, on the basis of similarity to other herpesvirus origins.     

The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) alpha subunit. Cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B

Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87% identity at the amino acid level) and to the rest of the integrin alpha subunits (38%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or "L" domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.     

Antigenic domain 1 is required for oligomerization of human cytomegalovirus glycoprotein B

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant virion envelope protein that has been shown to be essential for the infectivity of HCMV. HCMV gB is also one of the most immunogenic virus-encoded proteins, and a significant fraction of virus neutralizing antibodies are directed at gB. A linear domain of gB designated AD-1 (antigenic domain 1) represents a dominant antibody binding site on this protein. AD-1 from clinical isolates of HCMV exhibits little sequence variation, suggesting that AD-1 plays an essential role in gB structure or function. We investigated this possibility by examining the role of AD-1 in early steps of gB synthesis. Our results from studies using eukaryotic cells indicated that amino acid (aa) 635 of the gB sequence represented the carboxyl-terminal limit of this domain and that deletion of aa 560 to 640 of the gB sequence resulted in loss of AD-1 expression. AD-1 was shown to be required for oligomerization of gB. Mutation of cysteine at either position 573 or 610 in AD-1 resulted in loss of its reactivity with AD-1-specific monoclonal antibodies and gB oligomerization. Infectious virus could not be recovered from HCMV bacterial artificial chromosomes following introduction of these mutations into the HCMV genome, suggesting that AD-1 was an essential structural domain required for gB function in the replicative cycle of HCMV. Sequence alignment of AD-1 with homologous regions of gBs from other herpesviruses demonstrated significant relatedness, raising the possibility that this domain may contribute to multimerization of gBs in other herpesviruses.     

Structural and functional characterization of a cell surface binding protein of vaccinia virus

The nature of the interaction between the enveloped DNA-containing poxviruses and the surfaces of host cells as a first step in virus infection is not known. In this investigation we have identified and defined structural and functional properties of a 32-kDa protein of vaccinia virus. This protein is part of the virus envelope and binds to the cell surface of various cultured cells. The gene encoding the 32-kDa viral protein was mapped and sequenced. It was found to code a 35,426-Da protein with a large N-terminal domain with sequence homology to carbonic anhydrases and a C-terminal domain with sequences similar to those of the attachment glycoprotein VP7 of rotavirus and to transmembrane proteins. A potential cell surface binding domain was within the last 50 amino acid residues of the C terminus. The 32-kDa protein is basic, predicted pI 8.67, is synthesized at late times post-infection, may form dimers held by disulfide bonds at the single cysteine 262, and is apparently non-glycosylated. The 32-kDa protein is a vaccinia virus antigen, with predicted antigenic sites located near amino acids 108-110 (carbonic anhydrase domain) and 298-299 (transmembrane domain). Several lines of evidence suggest that the 32-kDa protein is needed for efficient virus replication in cultured cells but that in addition to this protein other viral proteins are involved in the process of virus entry into cells.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.     

Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4.

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.     

The Complete Primary Structure of Molluscan Shell Protein 1 (MSP-1), an Acidic Glycoprotein in the Shell Matrix of the Scallop Patinopecten yessoensis

The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa. The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp. Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that the regular arrangements of COO⁻ groups in the D domain side chains may be important for specific control of crystal growth. Received September 19, 2000; accepted February 9, 2001     

Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants.

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.