Surinder S. Saini
University of Guelph
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Featured researches published by Surinder S. Saini.
European Journal of Immunology | 1999
Surinder S. Saini; Brian Allore; Robert M. Jacobs; Azad Kaushik
We analyzed VDJ and VJ rearrangements in IgM‐secreting B lymphocytes from a cow infected with bovine leukemia virus (BLV). BLV causes expansion of CD5+ and IgM+ B lymphocytes regardless of antigen specificity. The data showed that single point mutations contribute to the diversification of IgM antibodies. The most striking observation, however, is that approximately 9 % of theVDJ rearrangement in IgM‐secreting B cells encode an exceptionally long third complementarity‐determining region of the heavy chain (CDR3H; 56 to 61 amino acids) with multiple cysteine residues. Such an exceptionally long CDR3H is the first ever to be documented for an antibody in a species. These VDJ rearrangements encode functional IgM antibodies as some of these show polyspecific reactivity. The presence of even‐numbered cysteine residues in the CDR3H may provide hitherto unknown configurational ability to the antigen combining site via intra‐CDR3H disulfide bridging. In addition, the VDJ rearrangements encoding exceptionally long CDR3H paired with either novel Vλ1 or Vx1x genes, earlier noted not to be expressed. Overall, these experiments provide evidence that somatic hypermutations and generation of an exceptionally long CDR3H contribute to the diversification of IgM antibodies in cattle.
Molecular Immunology | 1997
Surinder S. Saini; Wayne R. Hein; Azad Kaushik
In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.
Scandinavian Journal of Immunology | 2002
Surinder S. Saini; Azad Kaushik
Analysis of seven variable‐diversity‐joining (VDJ) gene rearrangements in B splenocytes from a 125‐day‐old bovine foetus revealed an extensive heavy‐chain complementarity‐determining region 3 (CDR3H) length variation (9–56 codons). Indeed, the global CDR3H size spectratyping of foetal VDJ rearrangements substantiated such an extensive heterogeneity and was comparable with that noted in peripheral B lymphocytes of adult cattle. These observations are in contrast to species such as humans with extensive germline combinatorial capability where shorter CDR3H length is noted early during B‐cell development. Exceptionally long CDR3H (as in adult cattle) was noted in two foetal VDJ rearrangements encoded by a single germline VH gene. Further, two VH genes (gl.110.20 and BF2B5) were preferentially expressed in the foetal VDJ rearrangements. The DH gene‐encoded CDR3H region of foetal VDJ rearrangements is remarkable for repetitive GGT (glycine) and TAT (tyrosine) codons that favour the recruitment of somatic hypermutations. It appears that closely related germline DH genes, preferentially used in the hydrophilic reading frame, encode varying CDR3H lengths early during B‐cell ontogeny in cattle. A comparison of germline and expressed VH genes, especially in the CDR1 and CDR2, confirms that somatic hypermutations contribute to immunoglobulin (Ig)M antibody diversification in cattle. The biased nucleotide base use and high occurrence of ‘hot‐spot’ triplet (AGPy; AG pyrimidine base) in the CDRs predisposes to somatic hypermutations. Overall, these observations suggest that extensive CDR3H length heterogeneity, including the generation of exceptionally long CDR3H (up to 56 amino acids), and somatic hypermutations contribute to IgM antibody diversification in cattle. The extensive CDR3H length heterogeneity early during the B‐cell development may compensate for constraints imposed on antibody diversification owing to the limited germline sequence diversity of genetic elements in cattle.
Molecular Immunology | 2003
Farbod Shojaei; Surinder S. Saini; Azad Kaushik
We demonstrated earlier the existence of an exceptionally long third complementarity-determining region of the heavy chain (CDR3H) (up to 61 amino acids (aa)), with multiple cysteine residues, in some functional IgM antibodies of cattle. To understand the origin of such a long CDR3H, we have now characterized the germline diversity gene (D(H)) of the cattle. A 2.3kb genomic DNA fragment hybridizing with a newly developed DNA probe to putative bovine D(H) gene sequences was isolated, cloned and its nucleotide sequence determined. Inspection of the nucleotide sequence led to identification of three bovine germline D(H) gene segments of varying size: 42bp (14 possible codons), 58bp (19 possible codons) and 148bp (49 possible codons). The characteristic repetitive GGT and TAT codons, remarkable in the CDR3H region of fetal VDJ rearrangements likely encoded by germline genes, are noted in two of the identified germline D(H) genes. These D(H) genes are preferentially expressed in the third reading frame to encode hydrophilic glycine and tyrosine residues in the CDR3H region. Phylogenetic analysis suggests that bovine D(H) genes are closest to rabbit and chicken D(H) genes. Thus, both short and long germline D(H) genes exist in cattle and these are capable of directly contributing to CDR3H size heterogeneity including the exceptionally long CDR3H region, apart from recombination associated mechanistic factors.
Veterinary Immunology and Immunopathology | 2002
Azad Kaushik; Farbod Shojaei; Surinder S. Saini
The bovine preimmune repertoire develops in the absence of maternal antibodies due to the placental barrier formed by syndesmochorial type of placenta. The limited germline sequence diversity, both at the heavy and light chain loci, imposes constraints on generation of combinatorial diversity in cattle. The cattle, thus, must employ other strategies for antibody diversification. Analysis of VDJ rearrangements in adult cattle have led identification of generation of large IgM antibody molecules that may have an exceptionally long CDR3H region (up to 61 amino acids). The IgM antibodies with an exceptionally long CDR3H are indeed functional as some of these recognize structurally dissimilar antigens. The antibody diversification in cattle involves generation of an exceptionally long CDR3H in addition to point somatic mutations.
Scandinavian Journal of Immunology | 2007
Surinder S. Saini; William Farrugia; Natarajan Muthusamy; Paul A. Ramsland; Azad Kaushik
Analysis of the heavy‐chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV‐1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1a (clone 2) and IgG1b (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216–230. The Thr224 and Thr226 of IgG1a were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1b were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three‐dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro–Ala–Ser–Ser indicated a potential structure‐enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O‐linked glycosylation sites and increasing the susceptibility to degradation by protease‐secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1d. Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1c that is distinct in both sequence and structure from the known sequence variants.
International Scholarly Research Notices | 2013
Surinder S. Saini; N. K. Maiti; Azad Kaushik
Partial characterization of immunoglobulin C gene of water buffalo (Bubalus bubalis) revealed high amino acid sequence identity with C of cattle (94.28%) and sheep (91.71%). Four amino acid replacements (Met-301, Val-310, Asn-331, and Thr-432) in C2, C3, and C4 of buffalo IgM are distinct, however. Unlike cattle, a codon deletion (GTG encoding valine at position 507 in cattle) and an insertion (GGC encoding glycine at position 532) occur in buffalo C4. Three N-linked glycosylation (Asn-X-Thr/Ser) sites (one at position 325–327 in C2; two at positions 372–374 and 394–396 in C3) differentiate buffalo IgM from cattle and sheep. Similar to cattle, buffalo IgM has fewer prolines in C2, which acts as hinge, which restricts Fab arm flexibility. Increased structural flexibility of the C1q-binding site in C3 compensates for the rigid buffalo C2 domain. Secondary structure of C1q-binding site is distinct in buffalo and cattle IgM where long alpha-helical structure is predominant that may be relevant to complement fixation function. Conserved protein motif “Thr-Cys-Thr-Val-Ala-His” provides protein signatures of C1q-binding region of ruminant species. The distinct structural features of C1q-binding site of buffalo and cattle IgM seem to be of functional significance and, therefore, useful in designing antibody based therapeutics.
International Immunology | 2003
Surinder S. Saini; William Farrugia; Paul A. Ramsland; Azad Kaushik
Veterinary Immunology and Immunopathology | 2010
Yfke Pasman; Surinder S. Saini; Elspeth Smith; Azad Kaushik
Veterinary Immunology and Immunopathology | 2009
Azad Kaushik; Marcus E. Kehrli; A. Kurtz; S. Ng; Madhuri Koti; Farbod Shojaei; Surinder S. Saini