Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan.
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.
Wang
Lauren W
LW
Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Leonhard-Melief
Christina
C
Haltiwanger
Robert S
RS
Apte
Suneel S
SS
D000071096:ADAMTS Proteins; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005643:Fucose; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012091:Repetitive Sequences, Nucleic Acid; D019700:Thrombospondin 1 / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
C454120:ADAMTSL1 protein, human; D016326:Extracellular Matrix Proteins; D019700:Thrombospondin 1; D005643:Fucose; D014364:Tryptophan; D000071096:ADAMTS Proteins; D008358:Mannose