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
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. C-mannosylation, , -, -, tryptophan
- 0. repeats, , ADAMTS-like 1, repeats, -
- 0. tryptophan, , -, C-mannosylation, tryptophan
- 1. attachment, , -, alpha-mannopyranose, tryptophan
- 10. mannosylated, , -, -, Trp residues
- 5. containing, , TSR1, O-fucosylation, -
- 5. containing, , TSR1, a Glc-Fuc disaccharide, -
- 7. TSR1, , TSR1, C-Mannosylation, -
Output(Part-Of) (sent_index,
protein,
site):
- 10. TSR1, Trp residues
- 5. TSR1, sequence
- 5. punctin-1, fragments
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):