The HLA class II or Ia molecules are mediators of the immune response, and have been associated with predisposition to certain diseases. Structural characterization indicates that human Ia molecules consist of a 34,000d Alpha chain glycoprotein associated non-covalently with a 29,000d Beta chain glycoprotein, both of which are encoded by the HLA complex. This Alpha, Beta heterodimer is associated with several """"""""invariant"""""""" chain glycoproteins creating an Ia """"""""oligomeric complex. We recently discovered that the Ia oligomeric"""""""" complex contains a previously undescribed 40-7-Kd chondroitin-sulfate proteoglycan component, and that the Ia Alpha and Beta chains can be sulfated. The broad objectives of this proposal are to structurally and biologically characterize the Ia associated proteoglycan component, and to elucidate the physiology of Alpha and Beta chain sulfation. We will radiolabel normal human lymphoid cells and lymphoblastoid cell lines with 35SO4 and 3H-amino acid precursors. The labeled molecules will be solubilized in non-ionic detergent, purified by anion-exchange chromatography, immunoprecipitated with anti-human Ia antibodies and analyzed by 1- and 2-D PAGE and sizing chromatography. The core-protein of the proteoglycan will be analyzed by tryptic peptic peptide mapping and partial amino acid sequencing. Using these techniques, we will determine the cellular distribution of the 40-70Kd proteoglycan in both Ia+ and Ia- cells of lymphoid and non-lymphoid tissues. We will determine the structural features of the proteoglycan component including the size of the core protein, the similarity, if any, of the core protein to known Ia glycoproteins, and the size and number of the glycosaminoglycan components. We will ascertain the fraction of Ia molecules associated with the proteoglycan, when and where the association takes place, the transit time of the proteoglycan to the cell surface, and the effect of proteoglycan synthesis inhibitors on the synthesis and plasma membrane expression of Ia. We will determine if removal or blockade of cell surface chondroitin sulfate interferes with binding during cellular interactions. The sulfate-bearing moiety on 35SO4-labeled Ia Alpha and Beta chains will be identified by pronase digestion, followed by HPLC and electrophoretic analyses. Because sulfation of Ia may play a role in cellular regulatory events, we will determine if reaction of Ia bearing cells with anti-Ia antibodies and mitogens alters sulfation of the Ia Alpha and Beta chains.
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