Human CD1d associates with prolyl-4-hydroxylase during its biosynthesis

Recent studies have shown that the CD1 family of proteins present various g lycolipid antigens to subsets of T cells. CD1d is expressed on human intest inal epithelial cells (IEC) and exists in two biochemical forms: 37-kDa, be ta2-microglobulin (beta 2m) independent, nonglycosylated, and 47-kDa, beta 2m dependent, glycosylated forms. The biosynthetic pathways and the mechani sms of generation of these two biochemically distinct forms of CD1d in huma n IEC are unknown. Using a human colonic cell line, T84, transfected with C D1d, the biosynthesis of CD1d was investigated. Pulse-chase metabolic label ing studies of T84 transfected with wild type CD1d demonstrated that CD1d w as a stable protein over a 4-day chase period. During the first 24 h of the chase, a novel 65-kDa glycoprotein was co-immunoprecipitated with CD1d. Mi crosequencing of this protein identified the glycoprotein as the alpha and beta subunits of the resident endoplasmic reticulum protein, prolyl-4-hydro xylase (P4H), an enzyme responsible for hydroxyl modification of proline re sidues. To study if either one or both biochemical forms of CD1d contained hydroxyproline residues, amino acid composition analysis of the 37 and 48 k Da was performed, and demonstrated that only the 37-kDa, but not the 48-kDa form of CD1d, contained hydroxyproline residues. These studies demonstrate that CD1d exhibits a prolonged association with P4H and that the 37-kDa fo rm contains hydroxyproline residues. This suggests that P4H association wit h CD1d during its biosynthesis results in a novel post-translational modifi cation of CD1d. (C) 2001 Elsevier Science Ltd. All rights reserved.