Biochemical characterisation of expressed bovine MHC class 1 molecules

Abstract

The bovine MHC (BoLA) class I region has been studied mostly by serology.

The products defined by the allo-antisera were thought to be alleles of a single highly polymorphic locus. However, studies involving the use of molecular biological and biochemical techniques revealed the presence of a second expressed locus.

Furthermore, the use of one dimensional isoelectric focussing reveals complicated patterns of molecules from each animal which indicates that the bovine class I encoding region is much more complex than previously thought. The aim of this project was to investigate the origins of the charge diversity of BoLA class I molecules observed using 1D-IEF.

The BoLA class I molecules appear to be glycosylated at a single N-linked position and that the carbohydrate moiety attached is of the complex type with up to three terminal sialic acid residues. The results also indicated that phosphorylation does not occur in class I molecules immunoprecipitated from resting bovine PBL. Furthermore, neither modification mentioned above is responsible for the observed charge heterogeneity. Additionally, 2D analysis of the immunoprecipitated samples suggested that alternative splicing is unlikely to be the major contributor to the observed complexity.

In order to investigate whether the different charge variants had different primary sequences, peptide mapping was employed. Different BoLA charge variants had distinct peptide maps. Animals sharing part of their serotype also show similarities in the digestion patterns they exhibited. These experiments suggested that each serological specificity is composed of a number of different polypeptides that represent the products of more than one class I locus. These results and further work involving the visual comparison of the 1D-IEF types of different animals obtained from immunoprecipitations with the MAb W6/32 and the different bovine allo-antisera suggested the existence of strong linkage disequilibrium among the class I molecules expressed by different loci in our herd. The number of different charge variants with different peptide maps indicated that the BoLA system has at least three expressed class I loci.

Additional studies involving the computer analysis of the available bovine class

I primary sequences revealed that the secondary structures of these sequences are similar to those of the molecules of the HLA system.

Furthermore, the number of residues in the transmembrane region (TM) of class I molecules in locus specific, the inspection of the BoLA sequences revealed them to cluster in three distinct groups with 37, 36 and 35 residues in their TMs respectively. This analysis gives support to the evidence produced in this thesis that at least three BoLA class I loci are present. However, these programmes with the available information did not allow us to identify locus specific structural motifs.

The significance and general implications of these findings are discussed.