Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active.

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aalpha, 66kDa; Bbeta, 55kDa; gamma, 48kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aalpha, Bbeta and gamma chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bbeta chain of the recombinant fibrinogen was N-glycosylated but the Aalpha chain, as in plasma fibrinogen, was not. The gamma chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated gamma chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.

Recombinant human antithrombin expressed in Chinese hamster ovary cells shows in vivo efficacy on rat DIC model similarly to plasma-derived antithrombin regardless of different N-glycosylation.

Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% alpha-isoform and 45% beta-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.

Production of recombinant human antithrombin by Pichia pastoris.

This paper deals with the production of recombinant human antithrombin (rAT) by the methylotrophic yeast Pichia pastoris. In preliminary methanol-limited fed-batch fermentation, the rAT concentration reached 324 mg/l at 192 h of cultivation, but the specific heparin cofactor (HC) activity of rAT in the culture supernatant was 10% of that of plasma-derived antithrombin (pAT). To improve the specific HC activity of rAT, effort was first focused on the optimization of culture pH and media composition, resulting in protection of rAT against pH-dependent instability and proteolytic degradation. However, even in the optimized methanol-limited fed-batch fermentation, the specific HC activity of rAT in the culture supernatant was still 20% that of pAT. To investigate the unknown mechanisms involved in the decreased specific HC activity of rAT, the culture supernatant of mock-transfected cells was prepared by methanol-limited fed-batch fermentation. When pAT was added to this supernatant, a rapid decrease in HC activity was observed; the residual HC activity was 26% after 24 h of incubation at 25 degrees C. The loss of pAT activity was prevented by addition of a formaldehyde scavenger, amino urea, to the supernatant. In addition, alcohol oxidase activity was observed in the supernatant, resulting in the accumulation of formaldehyde in the culture broth. These results suggest that the formaldehyde produced by methanol oxidation in the culture broth of P. pastoris might decrease the HC activity of rAT during fermentation. Replacing the methanol with glycerol as the carbon source improved the specific HC activity of rAT from 20% to above 40% of that of pAT. In the glycerol-limited fed-batch fermentation, rAT is expressed at 100 mg/l under the control of the truncated mutated AOX2 promoter.

Purification and characterization of recombinant human antithrombin containing the prelatent form in Chinese hamster ovary cells.

Antithrombin (AT) is a serine proteinase inhibitor and a major regulator of the blood coagulation cascade. AT in human plasma has two isoforms, a predominant alpha-isoform and a minor beta-isoform; the latter lacks N-glycosylation at Asn 135 and has a higher heparin affinity. From the difference in its folding states, the AT molecule can be separated into three forms: a native form, a denatured and inactive form known as the latent form, and a partially denatured form called the prelatent form. In this study, we purified and characterized recombinant human AT (rAT) containing the prelatent form produced by Chinese hamster ovary (CHO) cells. When rAT was purified at physiological pH, its specific activity was lower than that of plasma-derived human AT (pAT). The latent and prelatent forms were detected in rAT by using hydrophobic interaction chromatography analysis. However, when rAT was purified at alkaline pH, the prelatent form was reversibly folded to the native form and the inhibitory activity of rAT increased to a value similar to that of pAT. Highly purified rAT was analyzed and compared with pAT by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, amino acid composition, N-terminal sequence, monosaccharide composition, peptide mapping, and heparin-binding affinity. From these analyses, rAT was found to be structurally identical to pAT, except for carbohydrate side-chains. rAT in CHO cells had a high beta-isoform content and it caused a higher heparin affinity than by pAT and also pH-dependent reversible inhibitory activity.

Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides.

The yeast Pichia pastoris PNO1 (Phosphomannosylation of N-linked Oligosaccharides) gene, which is involved in phosphomannosylation of N-linked oligosaccharides, was cloned using the Saccharomyces cerevisiae MNN4 gene [Glycobiology 6 (1996) 805] as a probe. The PNO1 open reading frame (ORF) encodes a type II membrane protein composed of 777 amino acid residues. Only in the short region extending from amino acid position 450 to 606 of Pno1p, sequence homology to S. cerevisiae Mnn4p was observed at a level of 45%. The tandem repeat sequence of Lys-Lys-Lys-Lys-Glu-Glu-Glu-Glu characteristic of the C-terminal region of S. cerevisiae Mnn4p is not present in Pno1p. To investigate the function of the PNO1 gene, we constructed a PNO1 gene disruptant by replacement with an expression cassette of human antithrombin (AT), a glycoprotein in plasma. The cell growth and recombinant human antithrombin (rAT) production levels of the disruptant were similar to those of recombinant human antithrombin-expressing wild-type strains. Moreover, the level of alcian blue dye cell staining, which shows the presence of acidic sugar chains on the cell surface, was also similar. However, the phosphomannosylation ratio of N-linked oligosaccharides on recombinant human antithrombin decreased dramatically from 20% in wild-type strains to less than 1% in the PNO1 disruptant. When the PNO1 gene was re-introduced into the disruptant, the phosphomannosylation ratio recovered to the original level. These results suggest that the newly cloned PNO1 gene promotes phosphomannosylation only to core-like oligosaccharides, and not to the hypermannosylated outer chain, and that it has a different function from the MNN4 gene, which promotes the phosphomannosylation of both core and outer sugar chains.

Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris.

We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris. rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial. The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT). In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns. The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated. The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components. The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column. Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components. The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components. Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels.