Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells

Shutong Yang, David Nikodem, Eugene A. Davidson, D. Channe Gowda

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N-linked high mannose type structures and low levels of O-linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by ~14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove ~6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intracellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level. These results suggest that N-glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.

Original languageEnglish (US)
Pages (from-to)1347-1356
Number of pages10
JournalGlycobiology
Volume9
Issue number12
DOIs
StatePublished - Dec 1999

Fingerprint

Merozoite Surface Protein 1
Glycosylation
Plasmodium falciparum
Protein Sorting Signals
Cells
Peptides
Complementary DNA
Processing
Glycosylphosphatidylinositols
Vaccinia virus
Secretory Pathway
Mannose
Oligosaccharides
Viruses

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{9c7f465f47d745408cd8d26bb0935ae6,
title = "Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells",
abstract = "The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N-linked high mannose type structures and low levels of O-linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by ~14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove ~6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intracellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level. These results suggest that N-glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.",
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Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells. / Yang, Shutong; Nikodem, David; Davidson, Eugene A.; Gowda, D. Channe.

In: Glycobiology, Vol. 9, No. 12, 12.1999, p. 1347-1356.

Research output: Contribution to journalArticle

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T1 - Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells

AU - Yang, Shutong

AU - Nikodem, David

AU - Davidson, Eugene A.

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AB - The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N-linked high mannose type structures and low levels of O-linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by ~14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove ~6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intracellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level. These results suggest that N-glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.

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