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A specific increase in T cell extracellular acidification rate has been demonstrated recently when complexes of purified MHC class II molecules and antigenic peptides interact with T cell receptors (TCRs) on cloned T cells. The present study shows that such measurements of an increase in extracellular acidification rate can be used to evaluate the functional role of various N-linked oligosaccharides of MHC class II antigens. Affinity-purified murine IAk and IAs were deglycosylated in the presence of aspargine-amidase enzyme and were characterized by SDS-polyacrylamide gel electrophoresis. The complete removal of all three N-linked oligosaccharides from the alpha/beta heterodimer was confirmed by four different lectin-linked Western blot analyses. Similar to the native heterodimer, both deglycosylated IAk and deglycosylated IAs were fully capable of binding synthetic antigenic peptides derived from myelin basic protein (MBP). When equivalent amount of glycosylated and deglycosylated class II-peptide complexes were exposed to restricted cloned T cells, identical increases in T cell extracellular acidification rates were observed. The specificity of such increases in extracellular acidification rate was demonstrated by exposing cloned T cells to irrelevant complexes of glycosylated and deglycosylated class II and antigenic peptides. These results show how measurement of extracellular acidification rate can be used to study structure-function correlations of ligand-receptor interactions, and support an earlier observation that N-linked oligosaccharides of murine MHC class II molecules are not involved in either antigenic peptide binding or T cell recognition.

The MHC class II molecule is a heterodimeric glycoprotein consisting of one alpha and one beta polypeptide chain of almost identical molecular size. Recently it has been shown by others, and confirmed in our laboratory, that isolated monomers of murine MHC II molecules are capable of binding antigenic peptides like the alpha/beta intact heterodimer. In addition, preliminary results from our laboratory indicate that isolated single chain-peptide complexes of murine MHC class II molecules are capable of stimulating cloned T cells in an antigen specific manner. These results prompted us to isolate relatively large quantities of individual alpha and beta subunits of MHC II molecules for further in vitro and in vivo studies. Isolation of alpha and beta monomers proved to be difficult using conventional chromatographic methods. In this report we describe micro-preparative and preparative continuous flow electrophoresis methods by which milligram quantities of MHC II subunits can be purified. An optimal condition for the dissociation of heterodimeric MHC II into alpha and beta monomers was identified, and separation of human HLA DR2 and murine IAs monomers was accomplished. Both methods offer the resolving power of gel electrophoresis with the convenience of continuous sample elution. Purified MHC II subunits obtained by these methods were tested for their ability to bind antigenic peptides. Results presented in this study indicate that monomeric subunits of both human HLA-DR2 and murine IAs are equally active in specific binding of antigenic peptides like the native heterodimer.