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Glycobiology, 1999, Vol. 9, No. 12 1347-1356
© 1999 Oxford University Press

Glycosylation and proteolyticprocessing of 70 kDa C-terminal recombinant polypeptides of Plasmodiumfalciparum merozoite surface protein 1 expressed in mammaliancells

Shutong Yanga, David Nikodema, Eugene A.Davidson and D.Channe Gowdab

Department of Biochemistry and Molecular Biology, GeorgetownUniversity Medical Center, Washington, DC 20007, USA

The cDNAs that encode the 70 kDa C-terminal portionof Plasmodium falciparum merozoite surface protein1 (MSP-1), with or without an N-terminal signal peptide sequenceand C-terminal glycosylphosphatidylinositol (GPI) signal sequenceof MSP-1, were expressed in mammalian cell lines via recombinantvaccinia virus. The polypeptides were studied with respect to thenature of glycosylation, localization, and proteolytic processing.The polypeptides derived from the cDNAs that contained the N-terminalsignal peptide were modified with N-linked highmannose type structures and low levels of O-linkedoligosaccharides, whereas the polypeptides from the cDNAs that lackedthe signal peptide were not glycosylated. The GPI anchor moietyis either absent or present at a very low level in the polypeptide expressedfrom the cDNA that contained both the signal peptide and GPI signalsequences. Together, these data establish that whereas the signalpeptide of MSP-1 is functional, the GPI anchor signal is eithernonfunctional or poorly functional in mammalian cells. The polypeptidesexpressed from the cDNAs that contained the signal peptide wereproteo­lytically cleaved at their C-termini, whereas thepolypeptides expressed from the cDNAs that lacked the signal peptide wereuncleaved. While the polypeptide expressed from the cDNA containingboth the signal peptide and GPI anchor signal was truncated by ~14 kDa at the C-terminus, the polypeptidederived from the cDNA with only the signal peptide was processedto remove ~6 kDa, also from the C-terminus. Furthermore,the polypeptides derived from cDNAs that lacked the signal peptidewere exclusively localized intra­cellularly, the polypeptidesfrom cDNAs that contained the signal peptide were predominantlyintracellular, with low levels on the cell surface; none of thepolypeptides was secreted into the culture medium to a detectablelevel. These results suggest that N-glycosylationalone is not sufficient for the efficient extracellular transportof the recombinant MSP-1 polypeptides through the secretory pathway inmammalian cells.

a The authors Shutong Yang and DavidNikodem contributed equally to this study.

b Towhom correspondence should be addressed at: Department of Biochemistryand Molecular Biology, Georgetown University Medical Center, 3900Reservoir Road, NW, Washington, DC 20007


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