An original SERPINA3 gene cluster: elucidation of genomic organization and gene expression in the Bos taurus 21q24 region

Abstract

Background: The superfamily of serine proteinase inhibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, alpha1-antichymotrypsin, encoded by the SERPINA3 gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences.

Results: We provide additional information by analyzing the situation of SERPINA3 in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine SERPINA3 genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from SERPINA3-1 to SERPINA3-8. They share approximately 70% of identity with the human SERPINA3 homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution.

Conclusion: Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.

Figure 1

Southern blot analyses of bovine SERPINA3 organization. (A) Southern blot analysis of bovine genomic DNA digested with three different restriction enzymes NcoI, NciI and SacI. (B) Southern blot analysis of DNA from two overlapping BAC clones bI0123B08 and bI0511D06 digested with the same restriction endonucleases.

Figure 2

Genomic organization of bovine SERPINA3 genes. (A) General and schematic representation of SERPINA3 genes. Exons numbered from exon 1 to exon 5 are indicated by gray boxes, and intron sequences intron 1 to intron 4 by a thin line. (B) The Genbank accession numbers, the size in bases of each gene, mRNAs, exons and introns are listed in the table. The horizontal line in bold type separates the sub-group of the two other genes. The asterisk in intron 2 of SERPINA3-1 and SERPINA3-2 genes indicates the presence of the Bov-B LINE sequence.

Figure 3

Structure of the SERPINA3 locus on Bos taurus chromosome 21. The genes are labelled according to the classification in Figure 2B. Their names are indicated in bold characters and the names of loci established for Btau-2.0 genomic sequence assembly are added (when known) below between brackets. The different genes are localized on the four overlapping BAC clones covering approximately 235 Kb. The two question marks indicate the uncertainties of relative location for the genes SERPINA3-1/SERPINA3-2 and SERPINA3-3/SERPINA3-4, respectively. The SERPINA5 gene is also shown.

Figure 4

Multiple amino acid sequence alignment of the bovine SERPINA3 family. The eight putative bovSERPINA3 proteins were aligned using CLUSTALW [60]. Numberings refer to position in the alignment, above the sequences, and to the length of each sequence, at the end of the line. Dot indicates a common residue with that of the first sequence (bovSERPINA3-1), dash shows a gap, i.e. a position with an insertion-deletion event. Shaded characters indicate selectively conserved residues specific of the sub-group including bovSERPINA3-7 and bovSERPINA3-8. (▼) indicates asparagine (N) residue involved in a potential N-glycosylation site.

Figure 5

2D gel Western blot of a bovine SERPINA3 enriched fraction. High Mr fraction is eluted from a bovine diaphragma crude extract after gel filtration on a Sephadex G100 column, separated on 2D gel electrophoresis and hybridized with specific polyclonal antibodies. Arrows indicate the position of the two first characterized serpins bovSERPINA3-1 and bovSERPINA3-3. Note the linear alignment of spots corresponding to a different extent of phosphorylation of isoforms running with similar Mr on the second dimension. For more detail see the Methods section.

Figure 6

Phylogenetic trees including bovSERPINA3s. (A) BovSERPINA3 phylogeny based on Maximum likelihood (ML) method. The best ML tree (LnL = -3648.09) is rooted on porcine SERPINA3s. (B) SERPINA phylogeny based on ML method with the closest relatives of SERPINA3. The best ML tree (LnL = -16085.84) is unrooted. Bootstrap proportions are given for nodes supported in at least 50% cases. BP supporting clades are circled.