Enhanced Anti-Rotavirus Action of Human Cystatin C by Site-Specific Glycosylation in Yeast

Abstract

The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.

Genetically glycosylated ovomucoid third domain can modulate Immunoglobulin E antibody production and cytokine response in BALB/c mice

“…Post-translational modifications are known to have an impact on the IgE-binding activity [10]. The Pichia pastoris system has been used to study sitespecific glycosylations and enhancement of the molecular stability of recombinant proteins [11]. The number of mannose residues ('high-mannosylation' -glycosylation pattern) added to P. pastoris recombinant glycoproteins are mainly Man 8 GlcNAc 2 and Man 9 GlcNAc 2 .…”

Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site‐specific glycosylation in yeast

“…Both hcC and cC are nonglycosylated proteins. It had been reported that the anti‐rotavirus activity of hcC was substantially enhanced by site‐directed glycosylation using the yeast expression system (Nakamura et al 2004). Likewise, N ‐glycosylation at Asn106 greatly improved the freezing stability of recombinant cC over‐expressed in yeast Pichia.…”

Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization

“…Two synthetic genes encoding the primary amino acid sequences of HSA and HSB were prepared using overlap‐extension PCR , as illustrated in Fig. .…”