J E Sadler

Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine.

Elevated levels of plasma homocysteine are associated with both venous and arterial thrombosis. Homocysteine inhibits the function of thrombomodulin, an anticoagulant glycoprotein on the endothelial surface that serves as a cofactor for the activation of protein C by thrombin. The effects of homocysteine on thrombomodulin expression and protein C activation were investigated in cultured human umbilical vein endothelial cells and CV-1(18A) cells that express recombinant human thrombomodulin. Addition of 5 mM homocysteine to endothelial cells produced slight increases in thrombomodulin mRNA and thrombomodulin synthesis without affecting cell viability. In both cell types, thrombomodulin synthesized in the presence of homocysteine remained sensitive to digestion with endoglycosidase H and failed to appear on the cell surface, suggesting impaired transit along the secretory pathway. In a cell-free protein C activation assay, homocysteine irreversibly inactivated both thrombomodulin and protein C in a process that required free thiol groups and was inhibited by the oxidizing agents diamide or N-ethylmaleimide. By inhibiting both thrombomodulin surface expression and protein C activation, homocysteine may contribute to the development of thrombosis in patients with cystathionine beta-synthase deficiency.

Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a ≈47-kDa serine protease domain (light chain) and a disulfide-linked ≈120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (K m = 5.6 μm and k cat = 4.0 s−1) and by HL-BEK (K m = 5.6 μm and k cat = 2.2 s−1), but not by L-BEK (K m = 133 μm and k cat = 0.1 s−1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (K m = 0.27 mm and k cat = 0.07 s−1) and L-BEK (K m = 0.60 mm and k cat = 0.06 s−1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial K i = 99 nm and final K i * = 1.8 nm) over L-BEK (K i = 698 nm andK i * = 6.2 nm). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (K i * = 1.6 nm), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.

Molecular basis of von Willebrand disease type IIB. Candidate mutations cluster in one disulfide loop between proposed platelet glycoprotein Ib binding sequences.

Many variants of von Willebrand disease (vWD) with qualitatively abnormal von Willebrand factor (vWF) are recognized. In vWD type IIB, the abnormal protein displays enhanced affinity for a platelet vWF receptor, the glycoprotein Ib-IX complex. 14 patients from 7 unrelated families with vWD type IIB were studied to determine the molecular basis for this phenotype. Specific oligonucleotide primers were used to amplify portions of vWF exon 28 encoding a domain that interacts with the platelet glycoprotein Ib-IX complex. Candidate missense mutations were identified for all 14 patients by DNA sequencing, allele specific oligonucleotide hybridization, and restriction endonuclease digestion. These sequence changes occur in an 11 amino acid segment within a single disulfide loop bounded by Cys(509) and Cys(695). All of these sequence changes are C----T transitions within CG dinucleotides. Six patients from two unrelated families were heterozygous for the encoded sequence Arg(543)----Trp. Seven patients from four unrelated families were heterozygous for the encoded sequence Arg(545)----Cys; this sequence change appears to have occurred independently three times, once as a new spontaneous mutation. One patient with apparently sporadic vWD type IIB was heterozygous for the encoded sequence Val(553)----Met, and this appears to be a new mutation. None of these sequence changes was found in 100 normal alleles. These findings suggest that vWD type IIB may be caused by relatively few distinct mutations, that these mutations may cluster within a specific region of one disulfide loop in vWF domain A1, and that this region can modulate the affinity of vWF for the platelet glycoprotein Ib-IX complex.

Apical Sorting of Bovine Enteropeptidase Does Not Involve Detergent-resistant Association with Sphingolipid-Cholesterol Rafts

Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.