Superantigen properties of a human sialoprotein involved in gut-associated immunity.

Abstract

Protein Fv (pFv) is a recently described 175-kD gut-associated sialoprotein with a potent capacity for augmentation of antibody-dependent immune functions. To investigate the molecular basis for Fab-mediated binding of pFv, we evaluated a panel of 52 monoclonal IgM and found that -40% bound pFv. Whereas the majority (2 75% ) of VH3 and VH6IgM strongly bound pFv, only a small minority (< 20%) of IgM from other V H families bound pFv, and these antibodies had weaker binding interactions. Inhibition studies suggested that all binding occurred at the same (or overlapping) site(s) on pFv. Surface plasmon resonance studies demonstrated binding affinity constants up to 6.7 x 10 M-l for pFv. Biopanning of IgM and IgG Fab phage-display libraries with pFv preferentially selected for V H3 and V H6 antibodies, but also obtained certain V H4 1gM. V H sequence analyses of 36 pFv-binding antibodies revealed that binding did not correlate with CDR sequence, JH, or L chain usage. However, there was preferential selection of pFv binders with V H CDR3 of small size. These studies demonstrate that a protein which enhances immune defense in the gut has structural and functional properties similar to known superantigens. (J. Clin. Invest. 1995. 96:417-426.)

Repertoire cloning of lupus anti-DNA autoantibodies.

“…After informed consent, peripheral blood from these subjects was obtained as the source of mRNA for generation of IgG1 libraries, the predominant subclass of lupus anti-dsDNA Abs (12). As previously described (13)(14)(15)(16), PCR was used to rescue and clone Ab genes from human tissue into the phage display vector, pCOMB3H (gift of Dr. C.F. Barbas, The Scripps Research Institute, La Jolla, CA), to construct each library.…”

“…In a library, Ab genes are cloned within the phage-mid vector, and surface display phagemid clones are then selected based on Ab binding specificity. To construct the AA and BB libraries, a previously described panel of V H and V L first framework region specific primers (16) were supplemented with primers to ensure representation of newly reported V genes: VL1b/5, 5 Ј -CAG TCT GAG CTC ACT CAG CCA CC-3 Ј ; VL9, 5 Ј -CAG TCT GAG CTC ACT CAG CCT CCC-3 Ј ; VL3 Ј , 5 Ј -TCC TAT GAG CTC ACT CAG CCA CCC-3 Ј , VL8 Ј 5 Ј -CAG CTT GAG CTC ACT CAA TCG CCC-3 Ј ; VL7, 5 Ј -CAG GTT GAG CTC ACT CAA CCG CCC-3 Ј . The PCR conditions have previously been described (15,16).…”

“…To construct the AA and BB libraries, a previously described panel of V H and V L first framework region specific primers (16) were supplemented with primers to ensure representation of newly reported V genes: VL1b/5, 5 Ј -CAG TCT GAG CTC ACT CAG CCA CC-3 Ј ; VL9, 5 Ј -CAG TCT GAG CTC ACT CAG CCT CCC-3 Ј ; VL3 Ј , 5 Ј -TCC TAT GAG CTC ACT CAG CCA CCC-3 Ј , VL8 Ј 5 Ј -CAG CTT GAG CTC ACT CAA TCG CCC-3 Ј ; VL7, 5 Ј -CAG GTT GAG CTC ACT CAA CCG CCC-3 Ј . The PCR conditions have previously been described (15,16). In an effort to make comparable libraries, equal amounts of the 660-700 bp DNA band from each amplification was included in the AA and BB libraries.…”

“…The nucleotide sequences of the V H and V L regions were determined by automated sequencing (Applied Biosystems, Foster City, CA), and each was determined at least three times using two different primers with previously described primers (15,16). Analyses used Macvector 4.0 (IBI, New Haven, CT) and GCG (Madison, WI) software packages, with comparison to GENBANK and the V Base Directory.…”

“…To evaluate independently the molecular diversity in the AA and BB libraries, surveys were performed with V H gene family (16) and V gene-specific oligonucleotide probes (Table II). The IgG libraries from the twins had almost identical distribution of V H families.…”