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. 2014 Nov 24;9(11):e113650.
doi: 10.1371/journal.pone.0113650. eCollection 2014.

Identification of Transglutaminase Reactive Residues in Human Osteopontin and Their Role in Polymerization

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Free PMC article

Identification of Transglutaminase Reactive Residues in Human Osteopontin and Their Role in Polymerization

Brian Christensen et al. PLoS One. .
Free PMC article

Abstract

Osteopontin (OPN) is a highly posttranslationally modified protein present in several tissues where it is implicated in numerous physiological processes. OPN primarily exerts its functions through interaction with integrins via the Arg-Gly-Asp and Ser-Val-Val-Tyr-Gly-Leu-Arg sequences located in the N-terminal part of the protein. OPN can be polymerized by the cross-linking enzyme transglutaminase 2 (TG2), and polymerization has been shown to enhance the biological activity of OPN. However, little is known about the reactivity and location of the glutamine and lysine residues involved in the TG2-mediated modification of OPN. Here we show that TG2 catalyses the incorporation of 5-(Biotinamido)pentylamine at glutamines in both the N- and C-terminal parts of OPN, whereas TG2 primarily incorporated the glutamine-donor peptide biotinyl-TVQQEL-OH into the C-terminal part of OPN. By mass spectrometric analyses we identified Gln34, Gln42, Gln193 and Gln248 as the major TG2 reactive glutamines in OPN. The distribution of reactive Gln and Lys residues in OPN proved to be important, as the full-length protein but not the physiologically highly active integrin-binding N-terminal part of OPN were able to polymerize in a TG2-mediated reaction. Collectively, these data provide important new molecular knowledge about the mechanism of OPN polymerization.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. OPN contains TG2 reactive Gln and Lys residues.
(A) Schematic representation of OPN showing the N- and C-terminal parts of the protein, the thrombin cleavage site and the distribution of potential TG2 reactive Gln and Lys residues. The previously identified TG2 reactive Gln residues (Gln34, Gln36) and the integrin binding RGD sequence are indicated. (B–C) Maxisorp plates were coated with full-length OPN (triangle), the N-terminal part of OPN (circle) or the C-terminal part of OPN (square) (3 µg/ml) and subsequently incubated with increasing concentrations of TG2 in the presence of the amine donor 5-(Biotinamido)pentylamine (B) or the amine acceptor biotinyl-TVQQEL-OH (C). As negative control, wells not coated with OPN but incubated with 5-(Biotinamido)pentylamine or biotinyl-TVQQEL-OH and TG2 (5 µg/ml) are indicated with a cross. The samples were incubated for 3 h at 37°C. For all experiments data are expressed as mean ±S.D. (n = 3). The experiments were repeated four times.
Figure 2
Figure 2. Identification of TG2 reactive lysines by mass spectrometry.
(A) MALDI-MS of tryptic and chymotryptic peptides. Peptides observed with a mass corresponding to incorporation of biotin-TVQQEL are indicated with asterisks. (B) Table of tryptic and chymotryptic peptides containing biotin-TVQQEL. Location on the modified Lys residue in peptides containing more than one Lys was in some cases unambiguously achieved with the consideration that modified Lys residues are not trypsin substrates. The labeled Lys residues have been underlined and are shown in bold if the residue was unambiguously assigned. If the biotin label could not be assigned to a specific residue, the possible labeling sites are shown underlined and in italics. Monoisotopic molecular masses (MH+) were measured by MALDI-MS. The expected masses (MH+) include the biotinyl-TVQQEL-OH modification and were calculated using the GPMAW software. The ppm differences between the measured and expected masses are listed. Conservation shows how many of the 25 mammalian OPN sequences used in a multiple sequence alignment that contains the specific reactive Lys residue. Biotinyl-TVQQEL-OH labeling and MS analysis were repeated twice.
Figure 3
Figure 3. Identified TG2 reactive residues in OPN.
TG2-reactive glutamines (black) and lysines (grey) are highlighted. The major reactive Gln residues are indicated with numbers. The thrombin cleavage site is indicated with an arrow and the integrin binding RGD-sequence and the cryptic integrin binding site SVVYGLR are underlined.
Figure 4
Figure 4. Polymerization of full-length OPN and the N-terminal part of OPN.
The N-terminal part of recombinant OPN (A), full-length recombinant OPN (B–C) and human milk OPN (D) were incubated with TG2 in a 10∶1 ratio (w/w). In A and B, proteins were separated by 18% SDS-PAGE and stained with Coomassie Brilliant blue. In C and D, proteins were separated by 10% SDS-PAGE and detected by a polyclonal OPN antibody. A, B and D; OPN without TG2 (lane 1), or TG2-treated for 1 h (lane 2), 3 h (lane 3) and 16 h (lane 4). C, OPN without TG2 (lane 1), or TG2-treated for 15 min (lane 2), 30 min (lane 3), 1 h (lane 4), 3 h (lane 5) and 16 h (lane 6).

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Grant support

This work was supported by The Danish Council for Independent Research – Natural Sciences (grant No. 12-126168). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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