Student Theses and Dissertations

Date of Award

1978

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Bruce Cunningham

Keywords

H-2 antigens, histocompatibility, polymorphism, microglobulin, alloantisera, gene duplication.

Abstract

The major histocompatibility antigens are the predominant cell surface molecules recognized by the immune system in the rejection of grafts between individuals of the same species. The genes coding for the histocompatibility antigens are extremely polymorphic; the products of different alleles are defined serologically. The studies described in this thesis were carried out on the major histocompatibility (H-2) antigens of the mouse. The goals of these studies were to define the structure of the H-2 molecule in solution and on the cell surface and to compare the structures of different H-2 molecules to provide some insight into the nature of the genetic polymorphism. In order to carry out these studies, specific anti-H-2 alloantisera were produced. The sera recognized only one H-2 gene product as shown by antibody mediated cytotoxicity and immunoprecipitation assays. The alloantisera also recognized components of molecular weight 70,000-80,000 on mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp70) and are therefore closely related to or identical with that viral protein. Further experiments suggest that factors such as viral antigens on the immunizing cells, aging, and general stimulation of the immune system may contribute to the production of these antibodies in H-2 alloantisera. The physico-chemical properties of H-2Kb antigens solubilized by detergents and by the protease papain were compared by gel exclusion chromatography, ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detergent solubilized H-2 antigens (molecular weight 120,000) consist of two disulfide-linked heavy chains (47,000 daltons) and two noncovalently associated light chains (12,000 daltons). The light chain of histocompatibility antigens is known to be the protein β2-microglobulin. Alkylation with iodoacetamide prior to extraction prevented the formation of a disulfide linkage between the two heavy chains. A water-soluble 50,000-dalton molecule (Fs) consisting of a 39,000-dalton fragment (FH) of the heavy chain and one intact light chain was obtained by papain digestion of cells or detergent extracts. Experiments using the cross-linking reagent dimethyl-3,3'-dithiobispropionimidate indicated that the heavy and light chains of H-2 antigens are closely associated on the cell surface. Amino-acid sequence analysis of the FH fragment of H-2Kb by radiochemical techniques showed that it is identical to the detergent solubilized H-2Kb heavy chain in eight positions for the three amino acids tested. These data indicate that the fragment FH derives from the amino-terminus of the heavy chain and suggest that it projects outward from the cell surface, while the carboxyl-terminal region is associated with the plasma membrane. From these data a model is proposed for the H-2 antigens that includes the size and arrangement of the subunits on the cell surface and in solution. The H-2 heavy chains coded by the H-2K and H-2D genes of the haplotypes b, d and k were compared by SDS-PAGE, two dimensional gel electrophoresis and amino terminal amino acid sequence analysis. All of the H-2 gene products tested have similar overall structures. Furthermore, the amino terminal amino acid sequences of the heavy chains display considerable homology, supporting the hypothesis that the K and D genes of the major histocompatibility antigen complex have evolved by gene duplication. The amino acid sequences of the H-2 heavy chains are homologous to those reported for the heavy chains of the major histocompatibility antigens of man (HLA) and guinea pig (GPLA), indicating that the histocompatibility systems of mouse, man and guinea pig have a common evolutionary origin. The H-2 heavy chains also show distinct differences in molecular weight, isoelectric point and amino acid sequence. These differences appear to reflect the genetic polymorphism of the H-2 system. No feature has been defined, however, that unequivocally distinguishes allelic products of the K locus from those of the D locus. All of these findings have certain implications for the evolution and genetic organization of the murine histocompatibility antigens.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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