Immunoglobulin Light Chain Variable Region Research Articles

8] Direct complementary DNA cloning and screening of mutants using polymerase chain reaction

This chapter discusses the direct complementary DNA cloning and screening of mutants, using polymerase chain reaction. This chapter discusses the possibility of using the technique for direct complementary DNA (cDNA) cloning and for screening mutants for the variable region of immunoglobin heavy and light chains, establishing a new approach for cloning antibody cDNA. The chapter also discusses the revolutionary changes in molecular biology, resulting from the development of sequencing and polymerase chain reaction (PCR) techniques, have allowed to efficiently cloning the immunoglobulin heavy and light chain variable regions from hybridoma cells. The development of our reproducible experiments, combined with the knowledge of the immunoglobulin constant region sequences has made it possible to produce manmade antibodies without using conventional hybridoma techniques. The schematic summary of the method used to make mouse/human chimeric antibody is explained. Some important practical procedures should be considered when using PCR techniques for direct cDNA cloning. It is important to design appropriate primers for the PCR reaction. The chapter also discusses the developing a method to synthesize chimeric mouse/ human monoclonal antibodies by using synthetic long oligomers ligated together, containing just the mouse CDRs (complementarity-determining regions), and to synthesize mouse/human MAb containing fewer mouse sequences.

Rabbit antisera to the variable region domains of an anti- α(1 → 6)dextran using E. coli-produced V L and V H fusion proteins as immunogens

Bacteria were engineered for the expression of mouse immunoglobulin light chain variable region (V L) and heavy chain variable region (V H) fusion proteins. cDNAs encoding the V L and V H of anti- α(1 → 6)dextran hybridoma protein 19.22.1 were inserted into the pATH 10 prokaryotic expression vector downstream of trp operon sequences. V domains joined to approximately 330 amino acids of the trp E gene product encoded by the expression plasmids accumulated at high levels in E. coli. In addition, the V L domain was expressed with a 15 amino acid extension at low levels in lon mutant bacteria. The trp E-V L and trp E-V H proteins were used to raise antisera in rabbits and the V specificity of the sera demonstrated.

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

The expression of immunoglobulin heavy and light chain variable regions in the cytoplasm of Escherichia coli and formation of a functional heterodimer has been demonstrated. Variable domain sequences were taken from the heavy and light chain cDNAs of the monoclonal antibody Gloop 2 and engineered for expression in a dual origin expression vector. The engineered genes vhg2 and vlg2 were separately subcloned into the vector, creating two expression plasmids. Expression of the heavy and light chain variable region genes (encoding 116 and 109 amino acids respectively) was investigated in eight E. coli strains; the polypeptides were rapidly degraded in a host strain optimized for expression and in E. coli strains deficient in the major protease La (lon-). Accumulation was permitted in severely protease-deficient E. coli having a defective heat-shock response. A lon- mutation in this genetic background permitted even higher accumulation. Expression levels were 7 and 1% of total bacterial protein for light and heavy chain variable regions respectively. Expression of the heavy chain variable region gene was increased by including a longer Shine-Dalgarno sequence. Similar constructions in the light chain vector had no effect on expression levels. The insoluble variable region polypeptides were reconstituted into a heterodimer possessing the full antigen binding characteristics of both the parent monoclonal antibody and its Fab fragment.

A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen

The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.

Diversity and structure of human T-cell receptor beta-chain variable region genes.

The nucleotide sequences of 27 T-cell receptor beta cDNA clones isolated from a human peripheral lymphocyte library were determined and compared to five additional published sequences. These cDNA clones represent 22 distinct T-cell receptor beta-chain variable region (V beta) gene segment sequences, which fall into 15 different V beta gene subfamilies, each containing six or fewer members. From this analysis, we estimate that the repertoire of expressed human V beta genes is less than 59, apparently much smaller than the immunoglobulin heavy chain and light chain variable region (VH and VL) repertoires. Variability plots comparing these human V beta regions with each other and with published mouse V beta regions provide evidence for only four hypervariable regions homologous to those seen in comparisons of immunoglobulin V regions. Somatic hypermutation appears to be used infrequently, if at all, in these V beta genes.

Structural and functional implications of a restricted antibody response to a defined antigenic region on the influenza virus hemagglutinin.

A group of hybridoma antibodies that recognize structurally overlapping epitopes on the influenza virus hemagglutinin have been analyzed for the sequence of their immunoglobulin heavy and light chain variable regions. All VH regions derive from the same gene family, and only two Vk genes, from different families, are involved. The repetitive and restricted use of these variable region genes indicates that considerable structural requirements influence the generation of antibodies specific for this region of the hemagglutinin. The degree of amino acid variability which is permissive for interaction with this region suggests that two thirds of the possible replacement mutations may abolish either antibody function or specificity. Analysis of the somatic mutation which occurred in the individual antibodies indicates that the light chains acquired replacement mutations at the rate predicted for random mutation. The heavy chains, however, accumulated a 3-fold excess of replacement mutations over that predicted for random accumulation, correlating with the dominant role they apparently play in determining fine differences in the specificity of these antibodies. The effect of somatic mutation on the clonal amplification and diversification of these B cell lineages is discussed.

A new isotype sequence (V kappa 27) of the variable region of kappa-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides.

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.

Unrearranged immunoglobulin variable region genes have a functional promoter.

We have tested whether immunoglobulin light chain variable region genes are capable of directing initiation of transcription without undergoing the DNA rearrangement which creates a complete immunoglobulin gene. Two human Vk genes specifically initiated transcription in vitro at a site which is approximately 30 bases downstream of a TATA box and 20 bases upstream of the initiation codon. A Vk pseudogene which lacks a TATA box at an homologous position was not transcribed to a detectable extent in the in vitro system. One of the Vk genes was injected into Xenopus oocytes and it initiated transcription at precisely the same position as in the HeLa cell extract. It is suggested that the promoter which we have identified upstream of unrearranged Vk genes operates in lymphocytes after V-J joining has occurred to initiate transcription of light chain messenger RNA.

IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts.

An amino acid sequence analysis of the N-terminal immunoglobulin heavy and light chain variable regions (VH and VL) from 16 hybridoma proteins which bind phosphorylcholine as well as the complete sequence analysis of 9 of these VH regions is presented. There seem to be more VH regions participating in the phosphorylcholine response than can be encoded directly by germ-line VH gene segments. Moreover, the V regions from IgG antibodies are considerably more variable than those from their IgM counterparts. These observations raise the possibility that a somatic mechanism for V region diversification produces greater diversity in IgG than in IgM antibodies.

The specificity of T lymphocyte responses to chemically defined antigens.

A system is described that allows the definition of T cell receptor specificity with some precision. It involves immunization of guinea pigs with hapten coupled to mycobacteria. The T cells of such animals respond to many but not all carriers modified by that hapten. Such T cells recognize neither hapten nor carrier alone, but rather determinants involving both the hapten and the carrier. No evidence for hapten-specific T cells was found. A model of the antigen binding site of the T cell receptor emerged from these experiments. According to this model, the T cell receptor consists of a single site of relatively large extent involving multiple subsites which are of low and roughly equal affinity. Thus, the haptenic group is not immunodominant for T cells as it is for B cells and for anti-hapten antibody. This suggests that the antigen binding receptor on T cells differs in some fundamental way from that on B cells. It is proposed that antigen recognition by T cells is mediated by an immunoglobulin heavy chain variable region that is not paired with an immunoglobulin light chain variable region.

New, third class of amyloid fibril protein.

An unusual protein AR was isolated from the amyloid fibril preparation derived from a patient with primary amyloidosis. Protein AR was unique in its antigenicity, and revealed no structural identity with any known amyloid proteins or with immunoglobulin chains or fragments. Thus a new third class of amyloid fibril proteins besides the immunoglobulin light-chain variable region fragments and the nonimmunoglobulin protein AS, has been characterized. A component antigenically related to protein AR was found in the serum of the patient.