The transglutaminase activating metalloprotease inhibitor from Streptomyces mobaraensis is a glutamine and lysine donor substrate of the intrinsic transglutaminase

Abstract

Transglutaminase (TGase) from Streptomyces mobaraensis is an extra-cellular enzyme that cross-links proteins to high molecular weight aggregates. Screening for intrinsic substrates now revealed the dual Streptomyces subtilisin inhibitorlike inhibitor Streptomyces subtilisin and transglutaminase activating metalloprotease (TAMEP) inhibitor (SSTI), equally directed against subtilisin and the TGase activating metalloprotease TAMEP, is both a glutamine and a lysine donor protein.Reactivity of glutamines is lost during culture, most likely by TGase mediated deamidation, and, accordingly, cross-linking only occurred if SSTI from early cultures was used. Interestingly, release of buried endo-glutamines by the lipoamino acid N-lauroylsarcosine could restore SSTI reactivity. Formation of lipoamino acids by Streptomycetes suggests such compounds could also modulate in vivo TGase mediated SSTI cross-linking.

A Novel Transglutaminase Substrate fromStreptomyces mobaraensisTriggers Autolysis of Neutral Metalloproteases

“…Investigations were performed by TGase-mediated incorporation of biotinylated compounds and avidin-alkaline phosphatase staining on NC membranes. 8) Thus, mono-biotinylcadaverine (MBC, glutamine label) and 1-N-biotinyl-6-N 0 -(carbobenzoxy-L-glutaminylglycyl)diamidohexane (ZQGB, lysine label) were allowed to react distinctively with TGase-accessible glutamines and lysines respectively. Labeling of DAIP succeeded only in a few cases, suggesting severe modification of the inactivating protein by TGase during the culture of S. mobaraensis.…”

“…TGase-mediated hydrolysis of reactive SSTI glutamines during submerged culture of S. mobaraensis resulted in the discovery that lipoamino acids such as N-lauroylsarcosine can function as substrate modulators by expelling cryptic carboxamide groups from dipolar and hydrophobic interactions with the protein backbone. 8) Lipoamino acids have been reported to be formed by Streptomycetes when phosphates are exhausted. 10,11) Hence, phosphate deficiency after onset of aerial hyphae suggests, that lipoamino acids support protein cross-linking by TGase in the cell wall of S. mobaraensis.…”

“…6,8) SSTI appears to be a multi-functional protein, most likely regulating TAMEP and, indirectly, TGase activity, and protecting proteins against proteolytic degradation and heat inactivation. Accordingly, SSTI might be an important factor maintaining integrity of aerial hyphae.…”

“…After SDS-PAGE and protein blotting, labelled proteins were visualized using avidin-alkaline phosphatase/5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining, as described elsewhere. 8) GPC analysis. Size exclusion chromatography was performed using a 60-cm Superdex 75 column (Amersham Pharmacia, Freiburg, Germany) and 150 mM NaCl/2 mM CaCl 2 in 50 mM Tris/HCl pH 7.0 at a flow rate of 1 ml/min.…”

“…Labelling of DAIP was performed as described previously. 8) In brief, 2.3 mM DAIP and 0.13 mM ZQGB (or 2 mM MBC) in 0.1 M HEPES, pH 7.5, were incubated with 0.5 mM TGase from S. mobaraensis at 37 C for 2 h (final volume, 30 ml). The reaction was terminated by heating to 90 C in 30 ml of SDS buffer for 5 min.…”