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J D Lambris

Publications and source records attributed to J D Lambris.

17 recordsLinked to original sources
PubMed1992

Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

Animals↗
PubMed1992

Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the binding of CR1, factor H, and factor B.

CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Interaction between Epstein-Barr virus and a T cell line (HSB-2) via a receptor phenotypically distinct from complement receptor type 2.

Epstein-Barr virus (EBV), the causative agent of mononucleosis and several human cancers, infects cells via complement receptor type 2 (CR2, CD21) which also serves as the receptor for the third complement component, C3. Expression of this receptor is restricted to B lymphocytes, immature thymocytes, and certain epithelial cells. In the present investigation; we describe the presence of a seemingly novel EBV receptor which is phenotypically distinct from CR2. Among various leukemic T cells studied, one, HSB-2, demonstrates no reactivity to several anti-CR2 antibodies, yet it reacts strongly with EBV as detected by incubation with biotin-conjugated virus and streptavidin-phycoerythrin. The virus binding is specific as demonstrated by blocking with anti-EBV antibodies and with non-conjugated virus. Aggregated C3 also binds HSB-2 and is capable of partially inhibiting EBV binding. The absence of CR2 on HSB-2 is further supported by the lack of expression of specific mRNA, assessed by Northern blotting analysis and polymerase chain reaction. Viral internalization and infection is demonstrated with electron microscopy, with detection of EBV-DNA by Southern blotting, and with detection of EBNA-1 transcripts by the polymerase chain reaction. Even though HSB-2 does not express CR2, it nevertheless displays transcripts which have some homology to a CR2 cDNA probe under low stringency hybridization conditions. This probe encompasses approximately the N-terminal half of CR2 which includes the EBV-binding epitope(s). The HSB-2 message is 5.2 kb, a size distinct from the 4.7-kb message of B cell CR2s. In contrast, the 5.2-kb message in not seen, under similar hybridization conditions, with a probe comprising the C-terminal half of CR2. Collectively, the data indicate that a receptor molecule having distinct phenotypic characteristics from the known CR2 protein on B cells is utilized by EBV to target human T lymphocytes.

Animals↗
PubMed1991

Infection of human thymocytes by Epstein-Barr virus.

The Epstein-Barr Virus (EBV) causes infectious mononucleosis, and has been strongly associated with certain human cancers. The virus is thought to exclusively bind to B lymphocytes and epithelial cells via receptors (CR2/CD21) that also interact with fragments of the third component of complement (C3). Recent evidence, however, has challenged this belief. We have used two-color immunofluorescence analysis using biotin-conjugated EBV and streptavidin-phycoerythrin along with fluorescein-conjugated anti-T cell antibodies and demonstrated that CD1-positive, CD3-dull (immature) human thymocytes express functional EBV receptors. In four replicate experiments, the binding of EBV to thymocytes ranged between 8 and 18%. This interaction is specific as evidenced by inhibition with nonconjugated virus, anti-CR2 antibodies, aggregated C3, and an antibody to the gp350 viral glycoprotein that the virus uses to bind to CR2. EBV can infect the thymocytes as evaluated by the presence of episomal EBV-DNA in thymocytes that had been incubated with the virus as short as 12 days or as long as 6 weeks. Episomal DNA analysis was performed by Southern blotting with a EBV-DNA probe that hybridizes to the first internal reiteration of the viral DNA. The presence of the EBV genome is also supported by the detection of EBV nuclear antigen 1 in infected thymocytes, assessed by Western blotting with EBV-immune sera. The EBV infection is specific as determined by blocking experiments using anti-CR2 and anti-gp350 antibodies. Finally, virus infection of thymocytes can act synergistically along with interleukin 2 and induce a lymphokine-dependent cellular proliferation. In view of previously reported cases of EBV-positive human T cell lymphomas, the possibility is raised that EBV may be involved in cancers of T lymphocytes that have not been previously appreciated.

Antigens, CD19↗
PubMed1991

Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.

Amino Acid Sequence↗
PubMed1991

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules.

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (Mr 1000-4000) fragment from the alpha chain resulted in the generation of a Mr 36,000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the Mr 36,000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.

Amino Acid Sequence↗
PubMed1990

Neoantigens in complement component C3 as detected by monoclonal antibodies. Mapping of the recognized epitopes by synthetic peptides.

The different fragments of the third complement component, C3, generated upon complement activation/inactivation have the ability to bind to several other complement components and receptors as well as to proteins of foreign origin. These multiple reactivities of C3 fragments are associated with a series of conformational changes occurring in the C3 molecule during its degradation. The conformations acquired by the different C3 fragments are also associated with the exposure of neoantigenic epitopes that are specific for (a) particular fragment(s). In order to study these epitopes and thus the conformational changes occurring in C3, monoclonal antibodies (mAbs) recognizing such epitopes were produced in Balb/c mice after immunization with denatured human C3. Two of the three antibodies (7D84.1 and 7D264.6) presented in this study recognized predominantly surface-bound iC3b, and one mAb (7D323.1) recognized both surface-bound and fluid-phase iC3b. Although none of the mAbs recognized any other fluid-phase C3 fragment, all three antibodies detected micro-titre-plate-fixed C3b and iC3b, but not C3c or C3d. In addition to the reaction with human C3, mAb 7D323.1 also bound to micro-titre-plate-fixed rabbit C3. The epitopes recognized by the three mAbs were further localized by using synthetic peptides that were designed on the basis of the differential binding of the mAbs to the C3 fragments. All three antibodies reacted with C3-(924-965)-peptide, which represents the region of C3 between the kallikrein-cleavage site (923-924) and the elastase-cleavage site (965-966). On the basis of the binding of the mAbs to five different overlapping peptides spanning the region between residues 924 and 965 of the human C3 sequence, and the sequence similarity between human C3 and rabbit C3 within this area, the epitopes recognized by these antibodies are mapped. The contribution of the individual amino acid residues in the formation of the epitopes is discussed.

Antibodies, Monoclonal↗
PubMed1990

Monovalent ligands of complement receptor 2 inhibit whereas polyvalent ligands enhance anti-Ig-induced human B cell intracytoplasmic free calcium concentration.

We have performed experiments to investigate the role of ligands for complement receptor 2 (CR2) in human B cell activation. Flow microfluorimetry was used to assess changes in free intracytoplasmic calcium concentration [Ca2+] in indo-loaded B cells, immediately after exposure to anti-mu antibody and to monovalent or polyvalent CR2 ligands. As monovalent ligands we used the C3d fragment and synthetic C3 peptides (peptides P14, residues 1201-1214, and P28, residues 1187-1214). As polyvalent ligands we used i) an intact monoclonal mouse anti-CR2 antibody (HB5) and its F(ab')2 fragment, ii) tetravalent P13 [residues 1202-1214) 4-template), and iii) P28 conjugated to BSA (molar ratio 5/1). Anti-CR2 antibody HB5, tetravalent P13, and P28 conjugated to BSA, enhanced the ability of F(ab')2 fragments of the IgG fraction of goat anti-human mu antibody to increase human B cell [Ca2+]i. In contrast, the monomeric CR2 ligands C3d and P28 inhibited the anti-mu-induced increase in human B cell [Ca2+]i. Multivalent P13, P28, and the HB5, by themselves, did not affect B cell [Ca2+]i. These experiments suggest that the valence of the CR2 ligands is crucial for the nature (synergistic vs antagonistic) of the message transmitted through the CR2.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1990

Isolation and characterization of the third complement component of axolotl (Ambystoma mexicanum).

1. Using a monoclonal anti-human C3 antibody and a polyclonal anti-cobra venom factor antibody as probes, a protein homologous to the mammalian third complement component (C3) was purified from axolotl plasma and found to be axolotl C3. 2. Axolotl C3 consists of two polypeptide chains (Mr = 110,000 and 73,000) linked by disulfide bonds. An internal thiolester bond in the alpha chain was identified by the incorporation of [14C]methylamine and NH2-terminal sequence from the C3d fragment of C3. 3. Digestion of C3 by trypsin resulted in the cleavage of both the alpha and beta chains, generating fragments with a cleavage pattern similar to that of human C3. 4. The amino acid composition of axolotl C3 and the amino acid sequences of the thiolester site (and the surrounding amino acids), the cleavage site for the C3-convertase, and one of the factor I cleavage sites are similar to C3 from other vertebrates. 5. In contrast to human C3, which has concanavalin A binding carbohydrates on both the alpha and beta chains, only the beta chain of axolotl C3 contains such carbohydrates.

Ambystoma↗
PubMed1989

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis.

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.

Female↗
PubMed1989

The central segment of herpes simplex virus type 1 glycoprotein C (gC) is not involved in C3b binding: demonstration by using monoclonal antibodies and recombinant gC expressed in Escherichia coli.

Three monoclonal antibodies (MAbs) have been raised against cell membrane-derived herpes simplex virus type 1 glycoprotein C (gC-1). By using different DNA constructs of gC-1 expressed in Escherichia coli the sites recognized by these antibodies could be assigned to a peptide in the more hydrophobic and probably non-glycosylated middle third of the gC-1 molecule. This peptide segment corresponds to a 571 bp segment on the gC-1 gene located between the NcoI and the NruI restriction sites. None of the three MAbs interfered with the binding of the human serum complement component C3b to gC, which has been shown to be rather glycosylation-dependent. Since the data of other groups have suggested that antibodies directed against all regions of gC-1 inhibit C3b binding to gC-1, whereas our results suggest that the central part of gC-1 is not actually involved in C3b-binding activity, it can be inferred that the N- and C-terminal segments are involved in C3b binding by gC-1.

Animals↗
PubMed1989

Cell surface proteins reacting with activated complement components.

The biologic activities mediated by the products of complement activation include cellular, bacterial, and viral lysis, inflammation, phagocytosis, and immunoregulation. These responses are achieved through the interaction of the activated forms of several of the complement proteins with cell membrane proteins. This report reviews aspects of the structure, ligand specificity, and function of the various complement receptors with particular emphasis on those receptors which bind to the activated fragments of C3. In addition, we briefly summarize the surface proteins on foreign particles that bind C3 and their possible role in the pathogenesis of these organisms.

Animals↗
PubMed1985

Mapping of the C3d receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement.

The C3d domain of C3 contains the site that binds to the C3d receptor (CR2) which is expressed on B lymphocytes. It also contains a neoantigenic determinant that is recognized by monoclonal antibody (mAb) 130 and is expressed when C3b is cleaved to iC3b and subsequently to C3dg or C3d. mAb 130 inhibits the binding of C3d to CR2. In this study, the locations of the CR2-binding site and of the neoantigen recognized by mAb 130 within the C3d domain were investigated. Treatment of human C3d with CNBr generated two major fragments with Mrs of 12,500 and 8600. Binding studies showed that only the Mr 8600 fragment was capable of binding to both CR2 and mAb 130. Amino-terminal sequence analysis of the Mr 8600 fragment and comparison with the amino acid sequence derived from human C3 cDNA [de Bruijn, M. H. L. & Fey, G. H. (1985) Proc. Natl. Acad. Sci. USA 82, 708-712] placed it between residues 1199 and 1274 of the C3 sequence. Several peptides were synthesized according to the derived C3 sequence of amino acid residues 1209-1236. Based on their differential binding to CR2 and mAb 130, we localized the CR2-binding site and mAb 130 neoantigenic site, respectively, to residues 1227-1232 and 1217-1232 of the C3 sequence.

Amino Acid Sequence↗
PubMed1985

Antigenic relationship between the alpha-chain of C3, a leucocyte-surface antigen involved in the activation of phagocytic cells, and a 50,000 MW B-cell antigen.

A monoclonal antibody, M522, reacting with human monocytes, neutrophils and a proportion of non-adherent PBL in a pattern similar to OKM-1 and anti-Mo-1, and precipitating a dimer of MW 165,000 and 100,000 from neutrophils, was shown to react with C3 at an epitope localized on the alpha-chain of C3. F(ab)2-fragments of M522 stimulate the respiratory burst of neutrophils and monocytes. M522 differs from two monoclonal antibodies against the Mo-1 molecule with respect to the capacity to inhibit the binding of sheep erythrocytes coated with different C3-fragments to C-receptor carrying cells. It inhibited the binding of H-coated particles to B-lymphoid cells and precipitated a 50,000 MW molecule from RAJI cells and tonsil lymphocytes. The results obtained suggest an antigenic relationship between the alpha-chain of C3, the heavy chain of a membrane molecule involved in neutrophil/monocyte activation, and a B-cell molecule of MW 50,000.

Antibodies, Monoclonal↗
PubMedDate not supplied

Phylogeny of the third component of complement, C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in the C3 from different species.

The third component of complement, C3, binds to several other complement proteins. To study the diverse reactivities of C3, we analyzed the conservation of structural and functional features in the C3 from different species. First, we developed a method to purify swine (Po), rabbit (Rb), mouse (Mo), cobra (Co), Xenopus (Xe), axolotl (Ax), and trout (Tr) C3 from plasma. This involved protein precipitation by polyethylene glycol, followed by anion-exchange, gel filtration, and cation exchange chromatography. All C3's tested were comprised of two chains (alpha/beta-chain) and contain a thiolester bond within the alpha-chain. The two N-linked high-mannose carbohydrates found in human C3 were only conserved (as detected by ConA binding) in Rb C3. In contrast, Xe, Ax, and Tr C3 have this moiety only in the beta-chain and Po and Mo C3 only in the alpha-chain. Co C3, in contrast to cobra venom factor (CVF), lacks ConA binding carbohydrates in both chains. N-terminal amino acid sequence analysis of the alpha-, alpha'-, and beta-chains showed varying degrees of similarity within the different C3's. The N-termini of the Xe and Ax C3 beta-chains were found to be blocked. The conservation of binding sites in the different C3's for human complement receptors type one (CR1) and two (CR2) and for factors H and B was investigated due to the structural and functional similarities of these molecules and to the ability of some of them to bind to the same domain(s) in human C3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗