Title : Post-translational modification of
thrombospondin type-1 repeats in
ADAMTS-like 1 /
punctin-1 by
C-mannosylation of
tryptophan
Abstract :
- Protein C-mannosylation is the attachment of alpha-mannopyranose to tryptophan via a C-C linkage
- This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs)
- TSRs are especially numerous in and a defining feature of the ADAMTS superfamily
- We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1 / punctin-1 , which contains four TSRs (two with predicted C-mannosylation sites) , using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants
- Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the (36)WDAWGPWSECSRTC(49) sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation).
- Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp(39) and Trp(42)
- C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified
- Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-(3)H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells
- Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells
- Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency
- These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose.
- Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion