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

<div><p>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.</p></div

Transposon copy number in the RMCE-in HEK, HeLa, and murine ES cell clones.

<p><b>(A)</b> Southern blot of selected genomic DNA from <i>KpnI</i> digested HEK, (<b>B)</b> HeLa and <i>PstI</i> digested (<b>C)</b> mES clones. A 700bp dCTP³² labeled RFP probe (red rectangle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>) identified transposition of the gene cassette at bands > 2900bp as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>. Clone numbers are depicted above each lane.</p

Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System - Table 1

<p>Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System</p> - Table

Identified TG2 reactive residues in OPN.

<p>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.</p

Identification of major reactive Gln residues in OPN by MALDI-MS and MS/MS.

<p>Monoisotopic molecular masses (MH⁺) were measured by MALDI-MS. The expected masses (MH⁺) include the 5-(Biotinamido)pentylamine modification and were calculated using the GPMAW software. The ppm differences between the measured and expected masses are listed. 5-(Biotinamido)pentylamine labeling, peptide separation and MS analyses were repeated four times.</p><p>Identification of major reactive Gln residues in OPN by MALDI-MS and MS/MS.</p

OPN contains TG2 reactive Gln and Lys residues.

<p>(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.</p

Design of the RMCE docking site.

<p>(A) <i>Left</i>: Schematics of the commercially available Flp-in docking site. <i>Right</i>: Design of the SB transposon constituting the RMCE docking site present in the new RMCE-in cell lines. The RMCE docking site contains the CAG promoter which drives the expression of a RFP reporter linked to the puromycin-resistant gene through the ribosomal skip element E2A. (B) Schematic representation of donor plasmid used for Flp-in (left) and RMCE-in (right). The genetic element of interest (GEI) is represented by a GFP reporter. Note that the GFP in the RMCE-in donor does not contain a poly(A)-signal and utilizes the poly(A) from the RMCE docking site. The RMCE-in and the Flp-in donor plasmid are compatible with both the RMCE-in and Flp-in cell line. (C) Post recombination of the Flp-in system <i>left</i>: Prokaryotic elements, the initial marker and selection gene are present in the commercial Flp-in^™-293 cells post recombination, while the RMCE-in <i>right</i> leaves no prokaryotic DNA or initial reporter genes after cassette exchange.</p

Polymerization of full-length OPN and the N-terminal part of OPN.

<p>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).</p