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.
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- "Group A TSPs are C-mannosylated on the tryptophans of WXXW sequences in the properdin-like modules, as are other proteins with similar sequences , . The properdin-like modules also contain a CSX(S/T)C sequence that is O-fucosylated by O-fucosyltransferase 2, which then undergoes glucose addition, and is critical for protein maturation , , . O-glucosylation, as in a number of EGF-like modules containing a CXSXPC sequence between the first and second cysteines, occurs in the 1st EGF-like module (E1) in TSP-2  and presumably in TSP-1 and TSP-4. "
ABSTRACT: Thrombospondin-1 (TSP-1) is known to be subject to three unusual carbohydrate modifications: C-mannosylation, O-fucosylation, and O-glucosylation. We now describe a fourth: O-β-N-acetylglucosaminylation. Previously, O-β-N-acetylglucosamine (O-β-GlcNAc) was found on a threonine in the loop between the fifth and sixth cysteines of the 20(th) epidermal growth factor (EGF)-like module of Drosophila Notch. A BLAST search based on the Drosophila Notch loop sequence identified a number of human EGF-like modules that contain a similar sequence, including EGF-like module 1 of TSP-1 and its homolog, TSP-2. TSP-1, which has a potentially modifiable serine in the loop, reacted in immuno-blots with the CTD110.6 anti-O-GlcNAc antibody. Antibody reactivity was diminished by treatment of TSP-1 with β-N-acetylhexosaminidase. TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6. Analysis of tandem modules of TSP-1 localized reactivity of CTD110.6 to EGF-like module 1. Top-down mass spectrometric analysis of EGF-like module 1 demonstrated the expected modifications with glucose (+162 Da) and xylose (+132 Da) separately from modification with N-acetyl hexosamine (+203 Da). Mass spectrometric sequence analysis localized the +203-Da modification to Ser580 in the sequence (575)CPPGYSGNGIQC(586). These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.PLoS ONE 03/2012; 7(3):e32762. DOI:10.1371/journal.pone.0032762 · 3.23 Impact Factor
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- "Transient transfection of HEK293F cells (Invitrogen) and Cos-1 (ATCC, Manassas, VA) cells were performed in triplicate with 1 mg of the relevant ADAMTS10 full-length plasmid and 0.25 mg of plasmid encoding human IgG as previously described [Wang et al., 2007]. Cell lysate and serum-free conditioned medium were collected 48 hr posttransfection and electrophoresed on 6% "
ABSTRACT: We report the identification and functional analysis of the first missense ADAMTS10 mutation (c.73G>A; p.Ala25Thr) causing recessive Weill-Marchesani syndrome (WMS). The Ala25 residue affected by the missense mutation is at the -1 position relative to the ADAMTS10 signal peptidase cleavage site. p.Ala25Thr substituted full-length ADAMTS10 showed consistent and significantly diminished secretion in both HEK293F and Cos-1 cells. However, a C-terminally truncated construct lacking the ancillary domain and containing only the signal peptide, the propeptide and the catalytic domain (p.Ala25Thr Pro-Cat) was efficiently secreted in both HEK293F cells and Cos-1 cells. Edman degradation of purified p.Ala25Thr Pro-Cat and p.Ala25Thr substituted full-length ADAMTS10 from HEK293F cells demonstrated correct signal peptide processing. Thus, the p.Ala25Thr substitution hinders secretion of full-length ADAMTS10, but not Pro-Cat from cells, yet permits signal peptide removal. We infer that folding of the complex C-terminal ancillary domain is the rate-limiting step in biosynthesis of ADAMTS10, and that it (but not Pro-Cat) is sensitive to subtle changes in efficiency of signal peptide cleavage. These observations represent an unprecedented effect of a signal peptide mutation and support a model in which the initial cotranslational processing events during protein biosynthesis can have long-range effects on protein folding and secretion.Human Mutation 12/2008; 29(12):1425-34. DOI:10.1002/humu.20797 · 5.14 Impact Factor
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- "Two unusual forms of glycosylation are known to occur on TSRs. A homologue of Pofut1 known as protein O-fucosyltransferase 2 (Pofut2) (Luo et al., 2002, Luo et al., 2006a, Luo et al., 2006b) adds a fucose in α-linkage to a serine or threonine within TSRs containing the consensus sequence: C-X-X-[S/T]-C-X-X-G (between the first and second cysteines for class 1 TSRs, second and third for class 2) (Hofsteenge et al., 2001, Ricketts et al, 2007, Wang et al., 2007) (Figure 1D). This fucosylation event also appears to occur in the ER despite the lack of an obvious ER localization sequence in the Pofut2 sequence (Luo et al., 2006a). "
ABSTRACT: In the last two decades, our knowledge of the role of glycans in development and signal transduction has expanded enormously. While most work has focused on the importance of N-linked or mucin-type O-linked glycosylation, recent work has highlighted the importance of several more unusual forms of glycosylation that are the focus of this review. In particular, the ability of O-fucose glycans on the epidermal growth factor-like (EGF) repeats of Notch to modulate signaling places glycosylation alongside phosphorylation as a means to modulate protein-protein interactions and their resultant downstream signals. The recent discovery that O-glucose modification of Notch EGF repeats is also required for Notch function has further expanded the range of glycosylation events capable of modulating Notch signaling. The prominent role of Notch during development and in later cell-fate decisions underscores the importance of these modifications in human biology. The role of glycans in intercellular signaling events is only beginning to be understood and appears ready to expand into new areas with the discovery that thrombospondin type 1 repeats are also modified with O-fucose glycans. Finally, a rare form of glycosylation called C-mannosylation modifies tryptophans in some signaling competent molecules and may be a further layer of complexity in the field. We will review each of these areas focusing on the glycan structures produced, the consequence of their presence, and the enzymes responsible.The international journal of biochemistry & cell biology 11/2008; 41(5):1011-24. DOI:10.1016/j.biocel.2008.10.001 · 4.05 Impact Factor