PMID: 19671700

 

    Legend: Sugar

Title : Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1 / punctin-1 by C-mannosylation of tryptophan

Abstract :
  1. Protein C-mannosylation is the attachment of alpha-mannopyranose to tryptophan via a C-C linkage
  2. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs)
  3. TSRs are especially numerous in and a defining feature of the ADAMTS superfamily
  4. 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
  5. 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).
  6. 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)
  7. C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified
  8. 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
  9. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells
  10. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency
  11. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose.
  12. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion