The Effects of Cholesterol-3-sulfate (CH-3S) on the Phosphorylation of Human C3a (hC3a) <i>in Vitro</i> and on the Ability of hC3a to Induce Vascular Permeability in Rats

Abstract

The phosphorylation of human C3a (hC3a, anaphylatoxin) by two distinct protein kinases (PKA and CK-I) and the effect of cholesterol-3-sulfate (CH-3S) on this phosphorylation were biochemically investigated in vitro. It was found that (i) hC3a functions as a phosphate acceptor for PKA and CK-I, but not for CK-II; (ii) the CK-Imediated phosphorylation of hC3a requires the presence of 3 m mM CH-3S in a manner similar to the phosphorylation of HMG1 (CH-3S-binding protein) by CK-I; and (iii) CH-3S inhibits the PKA-mediated phosphorylation of hC3a in a dose-dependent manner (ID 50 ‫؍‬approx. 2 m mM). As expected, hC3a containing high levels of Arg-and Lys-residues stimulated approx. 3-fold CK-II activity (phosphorylation of a a-casein) in vitro. However, no significant effect of hC3a on CK-II activity was observed when hC3a was preincubated with CH-3S or fully phosphorylated by PKA in vitro. Furthermore, preincubation of hC3a with CH-3S diminished the ability of hC3a to induce vascular permeability in rats. The results provided here suggest that (i) hC3a is a CH-3S-binding protein; and (ii) CH-3S functions as a potent inhibitor for its physiological activities, including phosphorylation by PKA and CK-I, in vitro.

A Novel Consensus Phosphorylation Motif in Sulfatide- and Cholesterol-3-sulfate-Binding Protein Substrates for CK1 in Vitro

“…A Novel Phosphorylation Motif for CK1 in Several SCS-BP Substrates A computer database search and our previous reports [5][6][7][8][9] revealed that (i) several SCS-BPs (HMG1, PKCh, hC3a, FGF-BP, MBP and peptide M103, Table 1) contain a common consensus CK1 phosphorylation motif (K/R-X-K/R-X-X-S/T), which is different from the two canonical phosphorylation motifs [S(p)/T(p)-X-X-S/T and D/E-X-X-S/T] for CK1 3) ; and (ii) two functional proteins [human TP (an isoform, 441 amino acid residues) and human RhoA (196 amino acid residues)] possess multiple novel consensus phosphorylation sites for CK1. As expected, SCS highly stimulated the CK1d-mediated phosphorylation of TP (Fig.…”

“…[5][6][7][8][9] Briefly, three SCS-BPs (MBP, TP and RhoA) or five synthetic MBP peptides (approx. 2 mg each) were separately incubated for 30 min at 30°C in standard reaction mixtures (50 ml), comprising 40 mM Tris-HCl (pH 7.6), 2 mM DTT, 3 mM Mn 2ϩ , CK1d or other CK1 isoforms (approx.…”

“…32 P-Labeled SCS-BPs or MBP peptides in the reaction mixtures were detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, as described in our previous reports. [7][8][9] After incubation (for 30 min at 30°C), the phosphorylation of these protein substrates by CK1 was arrested by the addition of 0.5 ml 20% trichloroacetic acid (TCA) and 0.25 ml 0.1 M sodium pyrophosphate containing BSA (approx. 1 mg/ml) and 10 mM EDTA.…”

“…After drying, the 32 P-radioactivity on the membrane filter was measured with a liquid scintillation spectrometer, as described in our previous reports. [7][8][9] Two-Dimensional Phosphopeptide Mapping of 32 P-Labeled TP in Vitro After full phosphorylation of TP by CK1d with 5 mM [g-32 P]ATP (500 cpm/pmol) in the absence or presence of 3 mM sulfatide, the 32 P-labeled TP band was visualized by Coomassie brilliant blue (CBB) R-250 staining on the gel and the band corresponding to TP was excised, as described originally by Kurzer et al 11) Briefly, the gel slices containing 32 P-labeled TP were incubated overnight at room temperature in 1 ml of 50% acetonitrile containing 50 mM ammonium bicarbonate. The destained gel slices were incubated with 100% acetonitrile for 30 min at room temperature, and then dried by Speed-Vac.…”

Diabetes, glycaemic control and mortality risk in patients on haemodialysis: the Japan Dialysis Outcomes and Practice Pattern Study

“…More than 20 million people have diabetes in the USA, more than double the number 20 years ago, and 90-95% of cases are type 2 diabetes [1,2]. Furthermore, about 7.4 million people have diabetes in Japan [3].…”