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Modification of EGF-like module 1 of thrombospondin-1, an animal extracellular protein, by O-linked N-acetylglucosamine.

Hoffmann BR, Liu Y, Mosher DF - PLoS ONE (2012)

Bottom Line: 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.TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6.These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America.

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.

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MALDI-TOF/TOF sequencing of O-β-GlcNAc-modified tryptic peptide of the TSP-1 E1 module.The MALDI-TOF spectrum is indicated in (A) with both the −GlcNAc (2407.1-Da) and +GlcNAc (2610.2-Da) E1 module tryptic peptide peaks indicated. The MALDI-TOF/TOF y+1 ion series detected and spectrum are indicated in (B). Note the modification of Ser580 by O-β-GlcNAc from the difference between y13 and y14 ions. Also, note loss of a +203-Da adduct from a portion of the 2610.2-Da isolated peak and reversion to the unmodified peak as indicated.
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pone-0032762-g005: MALDI-TOF/TOF sequencing of O-β-GlcNAc-modified tryptic peptide of the TSP-1 E1 module.The MALDI-TOF spectrum is indicated in (A) with both the −GlcNAc (2407.1-Da) and +GlcNAc (2610.2-Da) E1 module tryptic peptide peaks indicated. The MALDI-TOF/TOF y+1 ion series detected and spectrum are indicated in (B). Note the modification of Ser580 by O-β-GlcNAc from the difference between y13 and y14 ions. Also, note loss of a +203-Da adduct from a portion of the 2610.2-Da isolated peak and reversion to the unmodified peak as indicated.

Mentions: To learn if the hypothesized recognition sequence is indeed the site of modification within the E1 module, MALDI-TOF/TOF MS analysis was performed on tryptic digests of reduced and alkylated recombinant E1 module. The CGACPPGYSGNGIQCTLELVPR tryptic peptide, which contains residues 572–587 (underlined) plus part of tail introduced by the expression strategy, was detected as +203-Da modified (2610.2-Da), along with a lesser amount of unmodified peptide (2407.1-Da) (Figure 5A). Isolation of the 2610.2-Da peak, followed by TOF/TOF sequence analysis to identify the site of modification localized the modification to Ser580 confirming O-β-GlcNAc modification (Figure 5B). During this process, some of the isolated 2610.2-Da peptide peak lost the modification, yielding one sequence with Ser580 modified and a second sequence lacking the adduct (Figure 5B).


Modification of EGF-like module 1 of thrombospondin-1, an animal extracellular protein, by O-linked N-acetylglucosamine.

Hoffmann BR, Liu Y, Mosher DF - PLoS ONE (2012)

MALDI-TOF/TOF sequencing of O-β-GlcNAc-modified tryptic peptide of the TSP-1 E1 module.The MALDI-TOF spectrum is indicated in (A) with both the −GlcNAc (2407.1-Da) and +GlcNAc (2610.2-Da) E1 module tryptic peptide peaks indicated. The MALDI-TOF/TOF y+1 ion series detected and spectrum are indicated in (B). Note the modification of Ser580 by O-β-GlcNAc from the difference between y13 and y14 ions. Also, note loss of a +203-Da adduct from a portion of the 2610.2-Da isolated peak and reversion to the unmodified peak as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293841&req=5

pone-0032762-g005: MALDI-TOF/TOF sequencing of O-β-GlcNAc-modified tryptic peptide of the TSP-1 E1 module.The MALDI-TOF spectrum is indicated in (A) with both the −GlcNAc (2407.1-Da) and +GlcNAc (2610.2-Da) E1 module tryptic peptide peaks indicated. The MALDI-TOF/TOF y+1 ion series detected and spectrum are indicated in (B). Note the modification of Ser580 by O-β-GlcNAc from the difference between y13 and y14 ions. Also, note loss of a +203-Da adduct from a portion of the 2610.2-Da isolated peak and reversion to the unmodified peak as indicated.
Mentions: To learn if the hypothesized recognition sequence is indeed the site of modification within the E1 module, MALDI-TOF/TOF MS analysis was performed on tryptic digests of reduced and alkylated recombinant E1 module. The CGACPPGYSGNGIQCTLELVPR tryptic peptide, which contains residues 572–587 (underlined) plus part of tail introduced by the expression strategy, was detected as +203-Da modified (2610.2-Da), along with a lesser amount of unmodified peptide (2407.1-Da) (Figure 5A). Isolation of the 2610.2-Da peak, followed by TOF/TOF sequence analysis to identify the site of modification localized the modification to Ser580 confirming O-β-GlcNAc modification (Figure 5B). During this process, some of the isolated 2610.2-Da peptide peak lost the modification, yielding one sequence with Ser580 modified and a second sequence lacking the adduct (Figure 5B).

Bottom Line: 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.TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6.These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America.

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.

Show MeSH