Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of Osteopontin

Christensen, Brian; Zachariae, Elias D.; Scavenius, Carsten; Kløverpris, Søren; Oxvig, Claus; Petersen, Steen V.; Enghild, Jan J.; Sørensen, Esben S.

doi:10.1021/acs.biochem.5b01153

Cited by 14 publications

(7 citation statements)

References 33 publications

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“…Human OPN cDNA had previously been cloned into the pcDNA3.1/ myc ‐His(−)A vector, 25 and human OPN without the signal peptide had previously been cloned into the bacterial expression vector pGEX‐6P‐2 26 …”

Section: Methodsmentioning

confidence: 99%

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FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin

Schytte

Christensen

Bregenov

et al. 2020

J of Cellular Biochemistry

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Osteopontin (OPN) is a ubiquitously expressed, multifunctional, and highly phosphorylated protein. OPN contains two neighboring integrin-binding motifs, RGD and SVVYGLR, which mediate interaction with cells. Phosphorylation and proteolytic processing affect the integrin-binding activities of OPN. Here we report that the kinase, FAM20C, phosphorylates Ser 146 in the 143 RGDSVVYGLR 152 motif of OPN and that Ser 146 is phosphorylated in vivo in human and bovine milk. Ser 146 is located right next to the RGD motif and close by the regulatory thrombin and plasmin cleavage sites in the OPN sequence. Phosphorylation of Ser 146 could potentially affect the proteolytic processing and the integrin-binding activities of OPN. We show that phosphorylation of Ser 146 does not affect the susceptibility of OPN for thrombin or plasmin cleavage. However, phosphorylation of Ser 146 significantly reduces the RGD-mediated interaction with the α v β 3 integrin in MDA-MB-435 and Moαv cells. This suggests a new mechanism by which specific phosphorylation of OPN can regulate interaction with the α v β 3 integrin and thereby affect OPN-cell interaction.

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Section: Methodsmentioning

confidence: 99%

“…Nonphosphorylated recombinant human OPN was expressed from a pGEX‐6P‐2 plasmid in E. coli BL‐21cc and subsequently purified as described by Christensen et al 26 OPN was purified from human milk 22 and bovine milk 27 as previously described.…”

Section: Methodsmentioning

confidence: 99%

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin

Schytte

Christensen

Bregenov

et al. 2020

J of Cellular Biochemistry

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“…Human OPN from breast milk was purified by anion-exchange chromatography on DEAE-Sepharose CL-6B (GE Healthcare, Uppsala, Sweden) and barium chloride and sodium citrate precipitation as described by Christensen et al (2010). Recombinant OPN was expressed in Escherichia coli BL21cc and purified as described by Christensen et al (2016).…”

Section: Purification and Deglycosylation Of Osteopontinmentioning

confidence: 99%

Milk osteopontin retains integrin-binding activity after in vitro gastrointestinal transit

Christensen

Karlsen

Jørgensen

et al. 2020

Journal of Dairy Science

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Osteopontin (OPN) is a multifunctional protein highly expressed in milk, where it is hypothesized to be involved in immunological signaling via the conserved Arg-Gly-Asp (RGD) integrin-binding sequence. Intervention studies have indicated beneficial effects of orally administered OPN in animal and human infants, but the mechanisms underlying these effects are not well described. To induce physiological effects, OPN must resist gastrointestinal transit in a bioactive form. In this study, we subjected bovine milk OPN to in vitro gastrointestinal transit, and characterized the generated fragments using monoclonal antibody and mass spectrometric analyses. We found that the fragment Trp 27 -Phe 151 containing the integrin-binding RGD sequence resisted in vitro gastric digestion. This resistance was dependent on glycosylation of threonine residues near the integrin-binding sequence in both human and bovine milk OPN. Furthermore, the fragment Trp 27 -Phe 151 retained the ability to interact with integrins in an RGD-dependent process. These results suggest a mechanism for how ingested milk OPN can induce physiological effects via integrin signaling in the intestine.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Lee

Salazar

Joseph

et al. 2019

Laboratory Investigation

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Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN mice underwent hindlimb ischemia surgery and 1.5 × 10 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

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Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of Osteopontin

Cited by 14 publications

References 33 publications

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin

Milk osteopontin retains integrin-binding activity after in vitro gastrointestinal transit

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

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