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

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.

OPN‐a induces muscle inflammation by increasing recruitment and activation of pro‐inflammatory macrophages

“…Transglutaminase 2 catalyses OPN polymerization at multiple Gln residues coded by exons 2–5 (Christensen et al . 2014). The deleted exons 5 (OPN‐b) and 4 (OPN‐c) contain one and three Gln residues, respectively, making the degree of isoform polymerization proportional to the number of transglutamination sites, with OPN‐a > OPN‐b > OPN‐c (Nishimichi et al .…”

“…The three spliced isoforms differ in protein cross-linking sites (Gln residues) that undergo covalent polymerization by transglutaminase 2, a difference that significantly alters OPN function (Higashikawa et al 2007;Nishimichi et al 2011). Transglutaminase 2 catalyses OPN polymerization at multiple Gln residues coded by exons 2-5 (Christensen et al 2014). The deleted exons 5 (OPN-b) and 4 (OPN-c) contain one and three Gln residues, respectively, making the degree of isoform polymerization proportional to the number of transglutamination sites, with OPN-a > OPN-b > OPN-c (Nishimichi et al 2011).…”

Osteopontin isoforms differentially promote arteriogenesis in response to ischemia via macrophage accumulation and survival

“…As pictured in Figure 1A, OPN-c (transcript variant 3) lacks exon 4, which contains several glutamine residues subject to transglutamination. 59,60 Consequently, unlike OPNa and OPNb, OPNc is likely unable to be cross-linked to form polymeric complexes, 61,62 which may in turn affect downstream functions. OPNc also lacks several phosphorylation sites, suggesting that differences in function may also stem from differences in post-translational modifications.…”

The Roles of Osteopontin in the Pathogenesis of West Nile Encephalitis

“…OPN can be polymerized to form poly OPN by enzyme transglutaminase-2 (TG-2) [ 34 ]. TG-2 is a Ca 2+ -dependent protein cross-linking enzyme that catalyzes the formation of a covalent, γ-glutamyl-ε-lysyl (isopeptide) bond between specific Lys and Gln residues of its substrate proteins [ 35 , 36 ].…”