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The methylotrophic yeast Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2. The AOX2 gene is transcribed at a much lower level than the AOX1 gene. Apart from this difference in expression levels, the two genes are regulated similarly. To study the role of cis-acting elements in the promoter region of the AOX2 gene, we constructed expression plasmids in which the human serum albumin (HSA) gene was placed under the control of various deleted or mutated AOX2 promoter derivatives. By analyzing the expression of HSA in P. pastoris transformants, we have identified three cis-acting regulatory elements in the AOX2 promoter. The positive cis-acting element AOX2-UAS, located between positions -337 and -313 (relative to the transcription initiation codon), is required for response to transcriptional induction by methanol in an orientation-independent manner, and artificial amplification of the AOX2-UAS resulted in an increase in the transcriptional activity of the promoter. A sequence homologous to AOX2-UAS was also found in the AOX1 promoter, and in methanol-regulated promoters in other methylotrophic yeast. Two negative cis-acting elements, AOX2-URS1 and AOX2-URS2 play a role in repressing transcription from the AOX2 promoter. The function of AOX2-UAS is completely repressed by this unique repression system when both the AOX2-URS1 and AOX2-URS2 are functional.

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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.

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.