Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 3, 2000, pp. 199

Biotecnología Aplicada, Volume 17, July-September 2000, p. 199

Primary Structure Analysis of Variable Regions Encoding ABI and AB2 Antibodies from the N-Glycolyl Ganglioside System

Alexis Pérez, Ana María Vázquez, Mauro Alfonso, Josefa Lombardero, Cristina Mateo, Geraudis Mustelier, Rolando Pérez

Center of Molecular Immunology. PO Box 16040, Havana 11600, CUBA. E-mail alexis@ict.cim.sld.cu

From a selection of papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.

Code Number: BA00063

Introduction

We have previously reported the generation of five anti-idiotype monoclonal antibodies against the N-glycolyl containing ganglioside-specific monoclonal antibody, P3. These AB2 monoclonal antibodies have been classified as AB2g according to its inability to induce antigen-independent responses against N-glycolyl containing gangliosides even when they all block ABI-AG interaction in a dose-dependent manner [1]. In this report we described the nucleotide and amino acid sequences from the heavy and light chains variable regions of P3, and two anti-idiotype antibodies, 1E10 and 3B11. Our data suggest a correlation between primary structures and immunochemical properties.

Materials and Methods

Total RNA was prepared from hybridoma cells obtained in our laboratory [1, 2]. First-strand cDNA synthesis was performed with primers specific for murine heavy and kappa chains constant regions. Variable regions were amplified by the polymerase chain reaction (PCR) using previous primers and framework 1 (FRW1)-specific primers, cloned in M13mp19 vector (Pharmacia) and sequenced by the dideoxy method (T7 sequencing kit, Pharmacia). Individual VH and VL sequences were searched against the EMBL/Genbank database for sequence homology with known murine genes. The JK and JH regions sequenced were compared with the five known murine JK genes and with the four known murine JH genes [3]. D region nucleotides were identified after alignment of CDR3H nucleotide sequences with known murine D minigenes [3].

Results and Discussion

VH P3 is a member of the Q52 (VIH 11) gene family. The most related sequence to VH P3 is a cDNA sequence of asws 1 (IgG2a, k), a mouse antibody generated in A.SW (H-2s) strain with specificity to U3 and U8 ribonucleoproteic particles [4]. There are only two nucleotide differences between P3 and asws 1 (codons 2 to 94), leading to only one amino acid replacement. Analysis of differences at both positions and comparison with previous reported sequences strongly suggest that VH P3 represent a new putative germline gene from the VH Q52 family. In CDR3, P3 has 14 amino acids and JH3 is used. The CDR3H of P3 MAb have a seven nucleotide-long D region from the DSP2 family. N nucleotide additions can be identified at both VH-D and D-JH junctions. VH P3 has an unusually extensive 3N region with 15 nucleotides. The high level of N nucleotides in this CDR3H explains its elevated content of infrequent amino acids. This feature could explain the strong immunogenicity of this MAb in syngeneic model, an aspect that distinguishes this MAb from others studied in our group [1]. The VH 1E10 is a member of the J558 family and is almost identical to that present in H35-C7, a hibridoma generated in Balb/c making antibody against the influenza virus hemagglutinin [5]. This strongly suggests that 1E10 and H35-C7 use the same germline VH gene. VH 1E10 has a 12 amino acid-long CDR3H, and its D region seems to result from recombination of DSP2.2 and DSP2 minigenes. The JH2 segment is used. The most related sequence to VH 3B11 appears in cDNA sequence of MRB9, a polyreactive murine monoclonal antibody of IgM isotype that binds to purified total histones [6]. Both sequences share a 95% homology with differences in 11 nucleotide positions (codons 1 to 94). Analysis of the distribution of replacement versus silent putative mutations and comparison with previous reported sequences shows that VH 3B11 has extensive somatic mutations. 3B11 has a 9 amino acid-long CDR3 and JH3 used.

Even when different VH gene segments contribute to the variable regions of 1E10 and 3B11, Ab2 shares a homologous sequence in the heavy chain CDR3 enriched in acidic residues. This sequence homology is based on homologous N nucleotide additions at VH-D junction and the use of the same D gene, DSP2.2, in the same reading frame. On the other hand, a higher level of basic than acidic amino acid residues was found in the CDRs of Ab1 P3. These findings suggest an involvement of residues in the motif shared by both Ab2 in the recognition of P3 idiotype.

References

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