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PubMed1989

Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR beta-1 chain and the Epstein-Barr virus glycoprotein gp110.

Rheumatoid arthritis is associated with the HLA antigen HLA-DR4. Disease susceptibility maps to the amino acid sequence QKRAA located in the third hypervariable region of the DR beta-1 chain. This region is thought to be a site of recognition for the T-cell antigen receptor. We searched for an antigen in the human environment that could induce T-cell recognition of this sequence. An analysis of protein and DNA databases revealed that the Epstein-Barr virus glycoprotein gp110, which is encoded by the BALF4 open reading frame, contains the sequence QKRAAQRAA, which is highly homologous to the rheumatoid arthritis susceptibility determinant. Experiments using antibodies to synthetic peptides showed that the QKRAA determinant is expressed on the gp110 protein. Humans with serologic evidence of Epstein-Barr virus infection had serum antibodies to gp110 and peripheral blood T cells that recognized peptides from gp110 and HLA-Dw4 encompassing the QKRAA determinant.

Amino Acid Sequence↗
PubMed1989

Development of biologically active peptides based on antibody structure.

Antibody molecules are composed of several functional domains, including a variable domain that contacts antigen and a constant domain. The hypervariable regions of antibody molecules play an integral role in determining their specificity. However, the delineation of specific residues most critical in binding is difficult. We have been studying a monoclonal antibody (87.92.6) that binds to the reovirus type 3 receptor on a number of cell types, down-modulates the receptor, and inhibits DNA synthesis in the cells. We have shown that a peptide derived from the second complementarity-determining region of the monoclonal antibody 87.92.6 light-chain variable region can reproduce both of these effects. We were also able to demonstrate specific amino acid residues and structural features involved in producing these effects. The study of antibody structure, coupled with molecular synthetic techniques, can lead to the development of biologically active substances with potential clinical use.

Amino Acid Sequence↗
PubMed1991

Humanized antibodies for antiviral therapy.

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

Amino Acid Sequence↗
PubMed1992

Evolution of major histocompatibility complex class II allelic diversity: direct descent in mice and humans.

The high degree of polymorphism seen at major histocompatibility complex (MHC) class II loci is a feature unique to the MHC. Most of the beta-chain polymorphism is localized in "hypervariable" regions (HVRs). HVR amino acid sequence similarity between distantly related species has recently been found. We have employed a Monte-Carlo statistic to show that shared HVR polymorphism between beta-chain genes of humans and mice represents direct descent of ancestral sequences rather than convergent evolution. Furthermore, half the sequence polymorphism seen in class II beta-chain genes of mice persists in evolution and is encoded by the same DNA sequence in humans. No evidence for increased mutation rate within the HVR was found. We postulate that the HVR can be considered the genetic unit of recombination, with selection for HVR sequences and combinations of HVRs constrained by functional considerations.

Alleles↗
PubMed1993

Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen.

The crystal structure of the Fab fragment of a human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody Fab has been determined at 2.8 A resolution in complex with a linear 16-residue peptide from the third hypervariable region (V3) of gp120. The first 9 residues of the peptide are ordered in the electron density maps, and their conformation is in partial agreement with the beta-strand-type II beta-turn structure predicted for this portion of the V3 loop. Notably, several of the peptide residues that are well conserved among different HIV-1 isolates contact a nonpolar 25-A-long groove in the antibody-combining site. The largely extended structure of the peptide differs from the beta-turns seen as the primary determinants in other published anti-peptide Fab structures. Analysis of the specific Fab-peptide interactions only partially explains the MN isolate specificity shown by this antibody.

Amino Acid Sequence↗
PubMed1995

Induction of antibodies to the human immunodeficiency virus type 1 by immunization of baboons with immunoglobulin molecules carrying the principal neutralizing determinant of the envelope protein.

The hypervariable region 3 (V3) within the disulfide-bridged loop of the envelope protein of the human immunodeficiency virus type 1 (HIV-1) contains an amino acid sequence that was defined as a principal neutralizing determinant (PND). A 19-amino acid residue consensus sequence (designated V3C) predicted from the PND sequences of 245 isolates as well as a sequence from the PND of the WMJ2 HIV-1 isolate (designated V3M) were expressed on the variable region of murine-human immunoglobulin (Ig) chimeras that were designated Ig-V3C and Ig-V3M, respectively. The HIV-1 sequences on the Ig chimeras preserved their antigenicity and interacted with antibodies specific for peptides encompassing the V3C and V3M sequences. In baboons, Ig-V3C and Ig-V3M induced antibodies that bound V3C and V3M peptides as well as the glycoprotein gp120 envelope protein of HIV-1 MN isolate. In addition, the baboons' antisera were able to prevent infection of CD4 SupT1 susceptible T cells by HIV-1 MN. Finally, Ig-V3M chimeras were able to stimulate in vitro production of antibodies specific for the HIV-1 envelope-derived peptides by lymphocytes from HIV-1-infected human subjects.

AIDS Vaccines↗
PubMed1995

Delineation of the minimal hepatitis B surface antigen-specific B- and T-cell epitope contained within an anti-idiotype-derived pentadecapeptide.

A pentadecapeptide (2F10 peptide) is capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg) at both the B- and the T-cell level. This peptide represents a sequence on the heavy-chain hypervariable region of a monoclonal "internal image" anti-idiotype (anti-id 2F10) that has partial sequence homology to the "a" determinant epitope of HBsAg. To identify the exact location of the B- and T-cell epitopes, four truncated peptides (peptides 1-4) were synthesized. Using these truncated peptides we have identified the minimal sequence (octapeptide 3) that represents a functional B- and T-cell epitope capable of generating HBsAg-specific antibodies and T cells. This to our knowledge represents the first example of a short peptide sequence functioning as both a B- and a T-cell epitope. We have also identified another T-cell epitope (2F10 peptide 4), but this peptide fails to elicit HBsAg-specific B cells and T cells. Thus, the 2F10 pentadecapeptide is composed of two nonoverlapping, functional T-cell epitopes only one of which is HBsAg specific. Since peptide 3 represents the complementarity-determining region and peptide 4 represents the framework region of the anti-id 2F10, we conclude that an 8-aa sequence from the complementarity-determining region of anti-id 2F10 is sufficient for the molecular mimicry of HBsAg. Finally, our experiments suggest that sequences flanking the minimal immunodominant epitope exert a considerable influence on the nature of antigenic processing that occurs and the resultant T-cell reactivity elicited.

Amino Acid Sequence↗
PubMed1996

Ancestral major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms.

Genes within the major histocompatibility complex (MHC) are characterized by extensive polymorphism within species and also by a remarkable conservation of contemporary human allelic sequences in evolutionarily distant primates. Mechanisms proposed to account for strict nucleotide conservation in the context of highly variable genes include the suggestion that intergenic exchange generates repeated sets of MHC DRB polymorphisms [Gyllensten, U. B., Sundvall, M. & Erlich, H. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3686-3690; Lundberg, A. S. & McDevitt, H. 0. (1992) Proc. Natl. Acad. Sci. USA 89, 6545-6549]. We analyzed over 50 primate MHC DRB sequences, and identified nucleotide elements within macaque and baboon DRB6-like sequences with deletions corresponding to specific exon 2 hypervariable regions, which encode a discrete alpha helical segment of the MHC antigen combining site. This precisely localized deletion provides direct evidence implicating segmental exchange of MHC-encoded DRB gene fragments as one of the evolutionary mechanisms both generating and maintaining MHC diversity. Intergenic exchange at this site may be fundamental to the diversification of immune protection in populations by permitting alteration in the specificity of the MHC that determines the repertoire of antigens bound.

Animals↗
PubMed1996

Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus.

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

Animals↗
PubMed2000

Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers.

The tremendous dynamics of HIV infection finds expression in the tempo of sequence diversification. Genetic diversity calculations require the clearance of a majority of infected cells, the obvious predator being anti-HIV immune responses. Indeed, infiltration of germinal centers (GCs) by HIV-specific CD8(+) cytotoxic T lymphocytes has been described. A corollary to this description would be limited diffusion of virus within lymphoid structures. HIV efficiently infects and replicates mainly in activated CD4(+) T lymphoblasts. These cells are found within GCs after their activation in the adjacent periarteriolar lymphoid sheath (PALS). Here GCs and PALS have been dissected from consecutive 10-micrometer sections through splenic tissue from three HIV-1-infected patients. Nested PCR amplification of the two first hypervariable regions of the env gene indicated that 38-78% of sections contained HIV-infected cells. Since there are several hundred CD4(+) T cells per GC section, approximately 0.09-0.64% harbor proviral DNA. Such a low frequency not only suggests that virions on the follicular dendritic cell surfaces do not readily infect adjacent T cells but also indicates highly restricted spread of HIV within GCs and the PALS. Sections were heavily infiltrated by CD8(+) cells, which, together with a large body of extant data, suggests that the majority of infected cells are destroyed by HIV-specific cytotoxic T lymphocytes before becoming productively infected. Finally, sequence analysis revealed that those HIV-positive cells were multiply infected, which helps explain widespread recombination despite a low overall frequency of infected cells.

Amino Acid Sequence↗
PubMed1995

Depalmitoylation of CAAX motif proteins. Protein structural determinants of palmitate turnover rate.

In the present study, we examined the effect of amino acid substitutions on the rate of turnover of palmitate bound to a model "CAAX" motif protein H-Ras. These experiments were designed to shed light on the specificity of the process that removes palmitate from prenylated proteins. H-Ras, protein A-Ras fusion constructs, and constructs with amino acid substitutions in the H-Ras hypervariable region were transfected into COS cells, and the turnover rate of palmitate bound to each expressed protein was measured. We found no evidence for strict sequence specificity for palmitate removal, but found a strong inverse correlation between palmitate turnover rate and the degree of membrane association for any given construct, with slower turnover rates associated with stronger membrane binding. These data support a model in which the palmitate turnover rate is determined by access to a depalmitoylating enzyme and argue against a more complex model in which specific recognition of palmitoylated proteins is required.

Amino Acid Sequence↗
PubMed1997

DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes.

Cytokines regulate cell growth by inducing the expression of specific target genes. We have recently identified a cytokine-inducible, immediate-early gene, DUB-1, that encodes a deubiquitinating enzyme with growth regulatory activity. In the current study, we have isolated a highly related gene, DUB-2, that is induced by interleukin-2. The DUB-2 mRNA was induced in T cells as an immediate-early gene and was rapidly down-regulated. Like DUB-1, the DUB-2 protein had deubiquitinating activity in vitro. When a conserved cysteine residue of DUB-2, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. DUB-1 and DUB-2 proteins are highly related throughout their primary amino acid sequence except for a hypervariable region at their COOH terminus. Moreover, the DUB genes co-localize to a region of mouse chromosome 7, suggesting that they arose by a tandem duplication of an ancestral DUB gene. Additional DUB genes co-localize to this region, suggesting a larger family of cytokine-inducible DUB enzymes. We propose that different cytokines induce specific DUB genes. Each induced DUB enzyme thereby regulates the degradation or the ubiquitination state of an unknown growth regulatory factor, resulting in a cytokine-specific growth response.

Animals↗
PubMed1997

Thermodynamic analysis of the interaction between the 0.5beta Fv fragment and the RP135 peptide antigen derived from the V3 loop of HIV-1 gp120.

The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.

Amino Acid Sequence↗
PubMed1998

Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase.

Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. We show here, however, that in vivo there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimicks that of Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized.

Amino Acid Sequence↗
PubMed1999

Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional characterization of its core domain loop.

The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain.

Amino Acid Sequence↗
PubMed1999

Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand.

Integrin alpha(V)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(V)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(V)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(V) integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha(V)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assays. alpha(V)beta(3) affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K(d) = 2.4 microM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K(d) = 80 nM), with no change in receptor number. In contrast, alpha(V)beta(3) in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta(3) cytoplasmic tails. Up-regulation of alpha(V)beta(3) affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(V)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

Adenoviridae↗
PubMed2002

Localization of the Rsr1/Bud1 GTPase involved in selection of a proper growth site in yeast.

Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.

Amino Acid Motifs↗
PubMed2002

Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes.

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.

Amino Acid Sequence↗