Conserved cysteines prevent C‐mannosylation of mucin Cys domains

Abstract

Mucins are major components of the mucus. Besides the highly O‐glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide‐forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C‐mannosylation, mucin CysDs have been suggested to be targets for C‐mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucin CysDs in Chinese hamster ovary (CHO) cells and analyzed the C‐mannosylation status. Mass spectrometric analysis revealed that the putative C‐mannose site is not or only barely C‐mannosylated. However, mutation of the adjacent cysteine residues enabled C‐mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C‐mannosylated, but introducing a cysteine at the −2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.