The ability of human VH3 immunoglobulins (Ig) to bind to staphylococcal protein A (SPA) via their Fab region is analogous to the binding of bacterial superantigens to T cell receptors. The present report establishes the structural basis for the interaction of SPA and VH3 Ig. We have studied a panel of 27 human monoclonal IgM that were derived from fetal B lymphocytes. As such, these IgM were expected to be encoded by unmutated germline genes. Binding to SPA in ELISA occurred with 15 of 15 VH3 IgM, but none of 12 IgM from the VH1, VH4, VH5, or VH6 families. The VH sequences of the 27 IgM were derived from 20 distinct VH elements, including 11 from the VH3 family. Use of D, JH, and CL genes was similar among VH3 and non-VH3 IgM. A comparison of the corresponding VH protein sequences, and those of previously studied IgM, identified a probable site for SPA binding that includes VH3 residues in framework region 3 (FR3), and perhaps FR1 and 3' complementary determining region 2. The results thus demonstrate that among human IgM, specificity for SPA is encoded by at least 11 different VH3 germline genes. Furthermore, like the T cell superantigens, SPA likely binds to residues in the VH framework region, outside the classical antigen-binding site of the hypervariable loops.
Monoclonal antibody (mAb) BEG-2 is a dsDNA binding IgM λ derived from a 12-week human fetus. Two binding site idiotypes (BEG-2 Id α and BEG-2 Id β) have been defined with the use of polyclonal rabbit anti-idiotypic anti-serum. BEG-2 Id α is located on the λ light chain and has been described previously. The BEG-2 Id β is present on the μ heavy chain. By means of a direct binding ELISA, BEG-2 Id β has been identified on EBV-derived mAbs from human fetal liver or spleen (5%), human cord blood (2.7%) and adult peripheral blood (1%). In addition, the Id is present on 8.5% of adult spleen-derived hybridoma antibodies and 6% of RA synovium-derived hybridoma antibodies. In all populations the presence of the Id is associated with binding to DNA and other polyanions. Competition indicated that the Id was located at or near the antigen-binding site on these molecules. To explore the structural basis of this binding, a major part of the BEG-2 chain was sequenced and found to be encoded by a member of the V H 4 family joined to a variant of J H 5 by a very short Diversity or N region. Of the BEG-2 Id β positive mAbs for which the V H family has been determined, five are encoded by V H 4 and two are encoded by V H 6, but none is encoded by other families. Thus, the BEG-2 Id β identifies a set of polyreactive antibodies that are common in fetal life, into adulthood and are encoded by V H 6 and, a subset of V H 4 genes.
In B cell precursors developing in fetal lymphopoietic tissue, the selection of VH, DH, and JH gene segments for initial H chain gene assembly is biased. The present study was designed to determine whether these biases persist in fully developed human fetal B cells and to examine specificities encoded by the favored elements. B cells were prepared from two sites representing different stages of development, i.e., fetal liver as a source of cells newly generated in that lymphopoietic environment and fetal spleen as a source of more mature cells, potentially subject to selective environmental factors. The expressed repertoires were sampled by two methods. EBV transformation so binding and structure could be examined simultaneously and generation of cDNA from individual, sorted, unstimulated B cells. We found that mature B cells in liver and secondary lymphoid tissue exhibit the same degree of bias in VH use we previously reported in lymphopoietic tissue of the same gestational age. However, the pattern of DH and JH use more nearly resembled that of the adult, suggesting that some constraints imposed by the rearrangement process are normalized rapidly. Sequences recovered from EBV-transformed clones were not distinguishable from transcripts recovered from single cells by direct amplification. Among antibodies expressed by the EBV clones, binding to self-Ag was common, binding profiles varied, and, in contrast to mice, there was little relationship between specificity and VH element. Interestingly, the two individuals studied differed in the VH element most commonly used. One resembled previously studied fetal repertoires in that VH56p1 encoded about 20% of expressed antibodies, whereas the other did not express VH56p1 and used VH26 in 25% of expressed antibodies. This was found to reflect a lack of the genomic VH56p1 allele, suggesting that genetic variation at the VH locus may significantly influence the emerging human antibody repertoire.
Alkaline phosphatase of matrix vesicles isolated vesicles from calcifying epiphyseal cartilage is a liverfrom fetal bovine epiphyseal cartilage was purified to kidney-bone isozyme.apparent homogeneity using monoclonal antibody affinity chromatography.The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Alkaline phosphatase of calcifying tissues has been impli-Tween 20 whereas the major Portion of nonspecific cated in the initial calcification of cartilage, bone, and dentine Protein W a s removed by this single Step.Of Various (1).Matrix vesicles which are enriched in alkaline phosphaagents tested, 0.6 M 2-amino-2-methyl-l-propanol, pH tase, Ca binding factors, and enzymes that degrade various 10.2, was the most effective in eluting 80-100% of the inhibitors of hydroxyapatite formation, are directly involved enzyme initially applied.Both Tween 20 and 2-aminoin the initiation of calcification in various tissues (for review 2 -m e t ~~l -l -~r o ~a n o l associated with the eluted ensee Ref.2).To fully understand the role of matrix vesicles in zyme were effectively removed by the sequential ap-calcification, each relevant component of the matrix vesicle plication of DEAE-cellulose and Sepharose CL-GB must be identified, purified, and characterized.Although the chromatography.Sodium dodecyl sulfate-polyacrylpurification and characterization of alkaline phosphatase of amide gel electrophoresis of the enzyme preparation matrix vesicles from chick and bovine epiphyseal cartilage treated with sodium dodecyl sulfate and mercaptoethhave been recently reported, several discrepancies with reanol showed the presence of a dominant band (using spect to its response to Mg2+ and its subunit molecular weight silver staining) corresponding to a molecular weight of have emerged (3, 4).These discrepancies could be due to 81,000.This molecular weight was nearer reported species difference, the use of different methods for vesicle values for rat liver (Ohkubo, A,, Langerman, N., and isolation (4), or other unknown factors.Furthermore, earlier Kaplan, M. M. (1974) J. Biol Chem.249,7174-7180) biochemical data indicate that a subunit molecular weight for and porcine kidney (Cathala, G., Brunel, C., Chappletmatrix vesicle alkaline phosphatase is not typical for a liver-Tordo, D., and Lazdunski, M. (1975) J. BioZ.Chem.kidney-bone type enzyme (3,4).To resolve these paradoxical 250, 6040-6045) alkaline phosphatase, than to pre-observations, and to provide amino acid composition data viously reported values for chicken (Cyboron, G .W., which have yet to be reported for bone or cartilage alkaline and Wuthier, R. E. (1981) J. Bioi.Chem.256, 7262-phosphatase, we purified the enzyme from fetal bovine matrix 7268) and fetal calf (Fortuna, R., Anderson, H. C., vesicles by specific monoclonal antibody affinity chromatog-Carty, R. P., and Sajdera, S. W. (1980) Calcif.Tissue raphy and Partially characterized the Purified enzyme.
Experimental animal models and observations in humans suggest that levels of Id and auto-anti-Id fluctuate reciprocally after Ag stimulation. In human monoclonal B cell disorders, however, the co-existence of paraprotein Id and its auto-anti-Id has been described in essential mixed cryoglobulinemia and in association with acquired C1 inhibitor deficiency. Because the majority of cryoglobulin IgM possess rheumatoid factor activity and thus bind the Fc region of IgG, we examined potential idiotypic interactions between cryoglobulin IgM and F(ab')2 fragments of autologous cryoglobulin IgG fractions. A rabbit antibody to the pepsin agglutinator site of human F(ab')2 was used as detection reagent. By recognizing epitopes exposed on F(ab')2 after the removal of Fc determinants by pepsin digestion, this reagent eliminates the detection of contaminating intact IgG. In a sensitive assay, we were unable to detect idiotypic interactions between the separated IgM and pepsin-digested IgG fractions of 10 mixed cryoglobulins. On the basis of these results, we suggest that in mixed cryoglobulinemia, the coexistence of paraprotein Id and its auto-anti-Id is unlikely.
The binding specificity of rheumatoid factors (RFs) to human Fc resembles that of some microbial Fc-binding proteins, suggesting conformational similarities in their Fc-binding regions. Using polyclonal chicken antibodies against SPA, we have detected a crossreactive determinant shared by human RFs from different individuals, but not by non-RF IgM and IgG. Chicken anti-SPA was shown to bind to 18 of 19 IgM RFs and 2 of 2 IgG RFs isolated from different individuals. This binding was inhibitable with SPA, fragment D of SPA, human IgG, and Fc fragment of IgG. The binding site for RF was located on the Fab' fragment of chicken anti-SPA. The antigenic mimicry of RFs by a protein of microbial origin suggests that the immune response to infectious agents could induce or modulate RF production through an internal image autoantiidiotype mechanism.
Work from our laboratories has shown that the major antigenic determinants for human rheumatoid factors (RFs) are in the Cγ2–Cγ3 interface region of IgG in the same area that binds staphylococcal protein A (SPA). Furthermore, the Fc binding proteins of groups A, C and G streptococci as well as the Fc binding proteins induced on cell surfaces by herpes simplex virus type I also bind to the Same area of IgG. These binding site similarities between RFs and the microbial Fc binding proteins suggested conformational similarities between the RF antigen combining regions and the Fc binding regions of the microbial proteins. This hypothesis was supported by the observation that antibodies to SPA bind to the antigen combining regions of most RFs as well as to the Fc binding region of the T15 group A streptococcal Fc binding protein. These findings indicate that RFs bear the conformational internal image of these microbial proteins and suggest that RFs could arise as antibodies to the idiotypic determinants on antibodies to microbial Fc binding proteins. Alternatively, microbial Fc binding proteins could present IgG to the immune system in a way that renders specific areas of the Cγ2-Cγ3 interface region immunogenic. These relationships between RFs and microbial Fc binding proteins may prove to be important for our understanding of the generation of RFs in rheumatoid arthritis.
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