Identification of a new component in the murine Ia molecular complex.

Abstract

In this report, we describe a previously unidentified component in the murine Ia antigen complex. SDS-PAGE analysis of anti-Ia immunoprecipitates prepared from spleen cells biosynthetically labeled with 35S-sulfate showed no detectable incorporation of 35SO4 into alpha, beta, or Ii chains but did not reveal the presence of a novel sulfate-bearing molecule of considerable molecular weight heterogeneity (46-69-kdaltons). The 46-69-kdalton molecule could be precipitated with monoclonal antibodies specific for I-A, I-E, and Ii glycoproteins but was not seen in control precipitates, nor in association with IgG or class I MHC molecules. Preliminary biochemical characterization indicated that the 46-69-kdalton product is extremely polydisperse, both in charge and apparent molecular weight, is sensitive to proteases, and bears the sulfate moiety on a large pronase-resistant structure. These results suggested this component might be a proteoglycan. Definitive identification of this component as a proteoglycan was accomplished by selective enzymatic degradation experiments which showed that the sulfate-bearing component of the 46-69-kdalton molecule is chondroitin 6-sulfate.

Identification of the glycosaminoglycan-attachment site of mouse invariant-chain proteoglycan core protein by site-directed mutagenesis.

“…This complex of surface-expressed processed antigen and Ia is bound by the T-cell antigen receptor, leading to helper-T-lymphocyte activation and release of soluble lymphokines from the T cell. The observations that Ii-CS is associated with Ia during terminal biosynthesis (9)(10)(11) and at the cell surface (11) and that interference with the synthesis of Ii-CS is accompanied by loss of antigen-presenting function (16,17) suggest some possible roles for Ii-CS in Ia biology. The Ii-CS molecule may enhance the binding between antigen-presenting cell and T cell, or it may affect Ia trafficking in ways that influence the association of Ia with processed antigen.…”

“…All three chains in the a/3li complex undergo coordinate posttranslational processing events. In addition to N-and O-linked oligosaccharides, some Ii chains receive the unusual addition of a chondroitin sulfate glycosaminoglycan (GAG) during Golgi processing (9,10), increasing their size to M, 45,000-70,000. Mr 31,000 Ii dissociates from the Ia heterodimer sometime between the trans-Golgi compartment and insertion of Ia into the plasma membrane, but some surface Ia continues to be associated with the GAG-bearing form of Ii (Ii-CS) (11).…”

“…After incubation for 24 hr in fresh medium, the cells were selected in medium containing mycophenolic acid (6 ug/ml), hypoxanthine (15 ,tg/ml), and xanthine (250 gg/ml). Clones (9). Lysates of radiolabeled or unlabeled cells were prepared and treated as previously noted (9).…”

“…Clones (9). Lysates of radiolabeled or unlabeled cells were prepared and treated as previously noted (9). Protein blotting from 10% acrylamide gels was performed (27), and Ii protein was identified on blots by means of the monoclonal anti-Ii reagent In-l. Two-dimensional electrophoretic analysis (28) was performed using a 3:1:1 ratio of pH 5-8, pH 8-9.5, and pH 3-10 Ampholines (LKB) for nonequilibrium pH-gradient electrophoresis (NEPHGE).…”

Identification of a sulfate-bearing molecule associated with HLA class II antigens.

“…Recently, in studies originally designed to examine the basis of murine Ia a-and -chain charge heterogeneity, we discovered a component unique to the Ia oligomeric complex (7). This component has biochemical characteristics suggesting that it is a proteoglycan.…”