Title :
O-fucosylation of thrombospondin type 1 repeats in
ADAMTS-like-1 /
punctin-1 regulates secretion: implications for the ADAMTS superfamily
Abstract :
- The ADAMTS superfamily contains several metalloproteases (ADAMTS proteases) as well as ADAMTS-like molecules that lack proteolytic activity
- Their common feature is the presence of one or more thrombospondin type-1 repeats (TSRs) within a characteristic modular organization
- ADAMTS like-1 / punctin-1 has four TSRs
- Previously, O-fucosylation on Ser or Thr mediated by the endoplasmic reticulum-localized enzyme protein-O-fucosyltransferase 2 ( POFUT2 ) was described for TSRs of thrombospondin-1 , properdin , and F-spondin within the sequence Cys-Xaa(1)-Xaa(2)-( Ser/Thr )- Cys-Xaa-Xaa- Gly (where the fucosylated residue is underlined)
- On mass spectrometric analysis of tryptic peptides from recombinant secreted human punctin-1 , the appropriate peptides from TSR2 , TSR3 , and TSR4 were found to bear either a fucose monosaccharide ( TSR3 , TSR4) or a fucose-glucose disaccharide ( TSR2 , TSR3 , TSR4)
- Although mass spectral analysis did not unambiguously identify the relevant peptide from TSR1 , metabolic labeling of cells expressing TSR1 and the cysteine-rich module led to incorporation of [(3)H]fucose into this construct
- Mutation of the putative modified Ser/Thr residues in TSR2 , TSR3 , and TSR4 led to significantly decreased levels of secreted punctin-1
- Similarly, expression of punctin-1 in Lec-13 cells that are deficient in conversion of GDP-mannose to GDP-fucose substantially decreased the levels of secreted protein , which were restored upon culture in the presence of exogenous l-fucose.
- In addition, mutation of the single N-linked oligosaccharide in punctin-1 led to decreased levels of secreted punctin-1
- Taken together, the data define a critical role for N-glycosylation and O-fucosylation in the biosynthesis of punctin-1
- From a broad perspective, these data suggest that O-fucosylation may be a widespread post-translational modification in members of the ADAMTS superfamily with possible regulatory consequences