Restricted usage of V_H and V_X genes by murine monoclonal antibodies against 3‐fucosyllactosamine

Abstract

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.

The improvement of an anti-CD22 immunotoxin

“…We exploited the alanine scan strategy to identify functionally important residues of the Fv. It is very likely that V H CDR3 region is the primary antigen binding site of HA22 Fv because V H CDR3 is the primary antigen binding site in most antibodies, 17,18 and because our previous studies using a phage-based in vitro evolution method 10,13 showed that Thr 100 -His 100a -Trp 100b in V H CDR3 is the only region whose modification drastically altered the affinity of HA22 Fv. Also, insertion of Gly before and after this region greatly reduced the activity of HA22 immunotoxin (unpublished data).…”

Groove-type Recognition of Chlamydiaceae-specific Lipopolysaccharide Antigen by a Family of Antibodies Possessing an Unusual Variable Heavy Chain N-Linked Glycan

“…Evidence of the highly evolved nature of the germ-line response to carbohydrate antigens and of the potential relationship between the V, D, and J gene segments is provided by the phenomenon of "V region restriction," where a given antigen will evoke a population of antibodies constructed from a small number of V genes combined with a number of different D and J genes (10,(12)(13)(14).…”

Heavy and light chain sequences of four monoclonal antibodies that bind galactosylgloboside (GalGb4)

“…It is interesting that V~4 4 1 is used to encode antibodies against a number of carbohydrate antigens, and we suggested previously [4] that the structure encoded by this VH gene segment may possess structural features that makes it particularly well adapted for anti-carbohydrate binding sites. The V~441-encoded anti-carbohydrate antibodies provide an interesting system for studying structurefunction relationships.…”

“…Total cellular RNA was prepared by the method of Chomczynski and Sacchi [12]. Sequencing was performed by a minor modification of the method of Geliebter et al [13], as previously described [4]. Additional sequence data were obtained by dideoxynucleotide sequencing [ 141 of single and doublestranded DNA templates, using Sequenase (US Biochemicals, Cleveland, OH) according to the manufacturer's instructions.…”

“…This antigen, which is also known as SSEA-3, is a developmentally regulated structure that is present in preimplantation mouse embryos up to the blastocyst stage [2,31. Although the structure 1 GSL used for immunization contains a terminal nonreducing 3-fucosyllactosamine (3-FL) sequence, which has elicited many mAb in BALBk mice [4], most of the antibodies elicited by structure 1 were directed against the internal GalGb, determinant. These antibodies bind with approximately equal avidity to structure 1 and GalGb,, which indicates that they have a "groove" type of binding site, i .…”