[Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

OBJECTIVE: To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. RESULTS: Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. CONCLUSION: We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.

Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS).

Hantaviruses cause an important human illness, HFRS. Blood samples from 22 HFRS-positive, six seronegative patients and 15 healthy controls were examined in 1995, during the largest HFRS epidemic in Croatia. Results of double- and triple-colour immunofluorescence analysis showed an increased percentage of cytotoxic T cells (CD3+CD8+) in seropositive patients compared with seronegatives and healthy controls. The majority of seropositive HFRS patients expressed activation and memory antigens on T and B lymphocytes. The percentage of CD23+ and CD21+ B lymphocytes was lower in seropositive patients. HFRS patients had elevated levels of sCD23 and five had elevated total IgE. The increased expression of both early and late T cell activation antigens, e.g. CD25, CD71 and HLA-DR, memory cells and sCD23 positively correlated with biochemical parameters (AST, ALT, urea, alpha2-globulin) during the acute phase of HFRS. The phenotypic changes observed, especially early and late T cell activation markers, as well as memory cells, could be useful parameters in the evaluation of HFRS course, and prognostic factors of HFRS severity. Additional attention should be paid to liver involvement in the pathogenesis of HFRS.

Ubiquitination and dimerization of complement receptor type 2 on sheep B cells.

Complement receptor type 2 (CR2) is a membrane-anchored glycoprotein that specifically binds C3d, as well as other ligands, and plays diverse roles in regulating immunity. Here we show that two distinct isoforms of CR2 are expressed on the surface of sheep B lymphocytes. One (CR2no 150 kDa) is structurally similar to known mammalian homologues while the other (CR2ub 190 kDa) has been modified by the covalent attachment of ubiquitin to the cytoplasmic domain and is identified for the first time. CR2no and CR2ub are expressed on the surface of sheep B cells as noncovalently associated dimers and the external topography of the two isoforms differs in some respect. The basis for these unusual higher-order structural properties may lie in the primary sequence of sheep CR2, since the transmembrane domain contains a region resembling a rare 7-amino acid dimerization motif, and two lysine residues in the cytoplasmic domain provide potential sites for posttranslational ubiquitination. The primary structures of sheep ubiquitin and C3d ligand are extensively conserved. In conjunction with the results of separate in vivo studies, these findings suggest that selective ubiquitination plays a role in modulating the higher-order structure and/or expression of CR2 during B cell development.

Characterization of C3-binding proteins on mouse neutrophils and platelets.

In the mouse, MCR1 and MCR2 on B lymphocytes are encoded by alternatively spliced Cr2 gene transcripts. Immune adherence receptors that bind C3 are present on mouse platelets and unstimulated neutrophils, yet they are not MCR1 or MCR2. To examine C3b- and C3d-binding proteins on mouse platelets and neutrophils, we performed C3b and C3d affinity chromatography as well as immunoprecipitation studies using previously described Ab to MCR1/MCR2 (mAb clones 8C12, 7G6, and 7E9 and polyclonal Ab BRN-1). Mouse neutrophils contained a 190-kDa membrane protein that specifically bound to C3b-Sepharose. Preabsorption of neutrophil proteins with anti-MCR1/MCR2 Ab did not affect the recovery of the 190-kDa C3b-binding protein by subsequent C3b affinity chromatography. Thus, this protein is immunologically distinct from the previously described MCR1 and MCR2 proteins. By virtue of its size and C3b-binding capacity, the 190-kDa protein was named C3bR-190. C3bR-190 was also apparent on platelets, but in reduced amounts. BRN-1 anti-MCR1/MCR2 Ab immunoprecipitated proteins of 125 and 150 kDa from surface-radiolabeled mouse platelets, which specifically bound to C3d-Sepharose. However, these proteins were not identified by mAb to MCR2, thus distinguishing them from previously described MCR2. These proteins were named C3dR-125 and C3dR-150. Therefore, we have identified a 190-kDa C3b-binding protein on mouse neutrophils and 125- and 150-kDa C3d-binding proteins on mouse platelets. These appear to be distinct from the heretofore identified mouse B lymphocyte MCR1 and MCR2. The identity of these C3b- and C3d-binding proteins on mouse neutrophils and platelets awaits further study.

Characterization of C3 receptors on cultured rat glomerular endothelial cells.

In this study we characterized C3 receptors on cultured rat glomerular endothelial cells (GEnC), using immunochemical and molecular techniques. GEnC membrane proteins were immunoprecipitated with a polyclonal antibody directed towards mouse complement receptor 2 (CR2). This anti-MCR2 immunoprecipitated GEnC proteins of 120 and 150 kDa. By immunohistochemistry, anti-MCR2 stained GEnC in rat glomeruli in vivo. Given the presence of CR2-like proteins on GEnC, subsequent studies were done to determine whether GEnC had C3-binding proteins. GEnC proteins of 80, 200, and 300 kDa specifically bound to columns of rat C3d-Sepharose and C3b-Sepharose, illustrating that these proteins were binding to the C3d portion of C3. The 80, 200, and 300 kDa C3d-binding proteins were distinct from the 120 and 150 kDa anti-MCR2 reactive proteins, as shown by immunoabsorption studies. Next, a specific cDNA probe for rat CR2 was generated by RT-PCR. Oligonucleotides were chosen from highly conserved regions in mouse and human CR2 spanning 224 bases, with the rationale that these would also be conserved in the rat. A 224 bp PCR product was generated from both rat GEnC and rat kidney cDNA, illustrating the presence of CR2 mRNA in these tissues. By Northern analysis, the CR2 PCR product hybridized to mRNA of 2 and 5 kb from GEnC. The 5 kb transcript was also identified in rat kidney mRNA. Therefore, proteins immunologically related to mouse CR2 are present in GEnC in vitro and in vivo. C3d-binding proteins of 80, 200, and 300 kDa are also present on rat GEnC, yet these appear to be immunologically distinct from the proteins identified by anti-MCR2. Whether the GEnC CR2 mRNA transcripts of 2 and 5 kb are translated into the 80 and 200 kDa C3d-binding proteins or the 120 and 150 kDa mouse CR2-like proteins remains to be defined.

Ligation of the functional domain of complement receptor type 2 (CR2, CD21) is relevant for complex formation in T cell lines.

We investigated the potential of CD21, the complement receptor type 2, to form receptor complexes with other membrane molecules on T cell lines. CD21 from T cell lines transformed with human T cell leukemia virus type I (MT2, HUT-102, C5.MJ, Mondi, and C91.PL) and T cell lines that were not virus transformed was analyzed by coprecipitation following cell lysis with digitonin. mAbs binding to functional and nonfunctional epitopes of CD21 and a polyclonal antiserum against its intracellular portion precipitated CD21, which was indistinguishable from CD21 on B cell lines. In contrast to B cells, where CD21 is complexed with CD19 and CD81 (target of anti-proliferative Ab 1) or, alternatively, with CD35 (CR1), no surface molecules could be coprecipitated with three of four mAbs from these T cell lines. Therefore, we assume that CD21 is not part of a preformed complex in T cell lines. OKB7, the only mAb directed against the functional C3d binding site, coprecipitated two proteins of 105 and 55 Mr with CD21 from MT2 and Mondi cells and from the T cell lines Jurkat E6-1 and SupT1. These bands were also recovered with CD21 precipitated from MT2 cells with C3d bound to Sepharose via the internal thioester, but were absent in CD21-expressing B cell lines. As C3d and OKB7 are functional ligands for B cells, we propose that upon ligation on T cells, CD21 associates with molecules of 105/55 Mr in the plasma membrane. Whether this is the first event of a signal delivered to the T cell is under current investigation.

Complex interaction between different proteinaceous components within the cell-wall structure of Candida albicans.

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface of Candida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to the C. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluorescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.

Characterization of rat complement receptors and regulatory proteins. CR2 and Crry are conserved, and the C3b receptor of neutrophils and platelets is distinct from CR1.

In the mouse, CR1 and CR2 on B lymphocytes are encoded by alternatively spliced Cr2 gene transcripts. Immune adherence receptors that bind C3b are present on mouse platelets and unstimulated neutrophils, but they are not CR1. In this study, rabbit anti-mouse CR1/CR2 Ab immunoprecipitated a 145- to 150-kDa CR2 protein from rat platelets, neutrophils, and splenocytes, but not a approximately 200-kDa CR1 protein. By Northern analysis, cDNA for mouse CR2 hybridized to mRNA of 3.7 and 5.2 kb from both mouse and rat splenocytes. The murine decay-accelerating factor and membrane cofactor protein analogue Crry was present in rat platelets, neutrophils, E, and splenocytes as two distinct proteins of 65 to 70 kDa and 75 to 85 kDa. Rat platelets, neutrophils, and splenocytes contained a novel 200-kDa cell membrane protein that specifically bound to a rat C3b-Sepharose column. We have named this protein C3bR-200. C3bR-200 was not identified by anti-mouse CR1/CR2 or anti-human CR1 Ab. Rat E lacked C3bR-200. Rat neutrophils and splenocytes also contained an 80-kDa C3b-binding protein that was distinct from Crry, which we have named C3bR-80. Therefore, CR2 and Crry are present in the rat, and have similar qualities to those from the mouse, except that CR2 is located on rat platelets and neutrophils, which is not the case in the mouse. Rat platelets, neutrophils, and splenocytes have a 200-kDa C3b-binding protein, C3bR-200, that is likely to be the rodent immune adherence receptor.

Ligand specificities of mouse complement receptor types 1 (CR1) and 2 (CR2) purified from spleen cells.

Murine complement receptor type 2 (MCR2) is homologous to human CR2, whereas murine CR1 (MCR1) is structurally and evolutionary different from human CR1. Since ligand specificities of MCR1 and MCR2 are not completely clarified, we analyzed their functional characteristics to correlate them with structural information obtained in molecular biological studies. MCR1 and MCR2 purified from spleen cells were incubated with thiol-Sepharose bearing murine C3b, iC3b, or C3d in the presence or absence of various anti-MCR1 or -MCR1/MCR2 mAbs. Bound and free MCR1 and MCR2 were quantitated by Western blotting or two-site immunoradiometric assay. MCR2 bound to C3d and iC3b similarly efficiently, and 5-fold less efficiently to C3b. These bindings were completely inhibited by MCR1/MCR2-crossreactive antibodies 7G6 and 4E3. MCR1 bound to C3b efficiently and this was partially inhibited by MCR1-monospecific antibody 8C12, but not by 7G6 and 4E3. Combinations of 8C12 and 7G6 or 4E3 completely inhibited MCR1 binding to C3b. Therefore, two binding sites, one unique to MCR1 and the other shared with MCR2, are involved in MCR1 binding to C3b. MCR1 bound also to C3d and this was completely inhibited by 7G6 and 4E3. The binding of this solubilized MCR1 to C3d was as efficient as that of MCR2 to C3d. It seems, therefore, that the site shared by MCR1 and MCR2 that is recognized by both 7G6 and 4E3 binds to C3d and less efficiently to C3b. These results clarify the ligand specificities of MCR1 and MCR2.