I GM degradation in b cells: proteolysis, direct visualization and auxiliary proteins

Y. Elkabetz, A. Kerem, O. Fingrut, M. Penner, D. Winitz, S. Bar-Nun

Research output: Contribution to journalArticlepeer-review

Abstract

Secretory IgM (slgM) is degraded in B cells at two distinct sites. In absence of light (L) chain, degradation occurs in the ER, while upon association with L chain, degradation is a post-ER event. Both ER and post-ER degradations were inhibited by ALLN, suggesting involvement, of a cystein protease. Yet, based on similar sensitivities to ALLN and ALLM and additive effects of ALLN and cycloheximide, proteolysis was carried out neither by proteasome nor by a short-lived protein implicated in post-ER degradation.In addition, we could not detect any ubiquitin-conjugated IgM molecules. Moreover, the in vitro degradation neither required added ATP nor was sensitive to apyrase. Nevertheless, inhibition by ATPγS suggested a role for lumenal ATP.Targeting to post-ER degradation can be attributed to the unique C-terminus of ps, the μstp. Hence, a TPO-μs,v was constructed by fusing μstp to tile C-terminus of human thyroid peroxidase. Upon transfection, an attempt is made to directly visualize this novel site by EM in various cell types. In order to isolate auxiliary proteins participating in slgM sorting, we first defined favoring conditions.In fact, the relatively oxidizing thiol and high Ca2+ concentration in the ER lumen contributed to the sorting fidelity. A 20kDa/,s-associated protein (p20) was isolated by cross-linking and co-immunoprecipitation. Indeed, this association was hampered by -mercaptoethanol or EGTA. Furthermore, the specific interaction of p20 with μs and with μsty in particular was indicated by anti-pstv antibodies recognizing ps but not /m and by specifically competing synthetic peptide representing/zst.

Original languageEnglish
Pages (from-to)A930
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997

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