Abstract
Secretory human interleukin 4 (hIL4) is an N-glycosylated pleiotropic cytokine. It is unknown if these N-linked glycans are required and essential for hIL4 protein stability, expression, secretion, and activity in vivo, and hIL4 expressed from Pichia pastoris yeast has not been tested to date. In this study, we successfully expressed human hIL4 in P. pastoris, the methylotrophic yeast, with a yield of 15.0mg/L. Using the site-directed mutagenesis technique, we made two mutant hIL4 cDNA clones (N38A and N105L) and subsequently expressed them in P. pastoris to analyze the relevant function of each N-glycosylation site on hIL4. Our results demonstrate that the glycosylation only occurs at position Asn38, but not Asn105. The glycosylated form of hIL4 unexpectedly has lower biological activity and lower stability when compared to its non-glycosylated form. The implications of this are discussed.
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