The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

TY - JOUR

T1 - The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

AU - Nagar, Rupa

AU - Garcia Castillo, Stefano S.

AU - Pinzon-Ortiz, Maria

AU - Patray, Sharon

AU - Coppi, Alida

AU - Kanatani, Sachie

AU - Mortiz, Robert L.

AU - Swearingen, Kristian E.

AU - Ferguson, Michael A. J.

AU - Sinnis, Photini

N1 - Copyright: © 2024 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.

PY - 2024/8

Y1 - 2024/8

N2 - Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

AB - Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

KW - Glycosylphosphatidylinositol

KW - Plasmodium

KW - malaria

KW - sporozoite

KW - circumsporozoite protein (CSP)

KW - metabolic labeling

KW - GC-MS

KW - glycosylphosphatidylinositol

UR - https://www.scopus.com/pages/publications/85200643272

U2 - 10.1016/j.jbc.2024.107557

DO - 10.1016/j.jbc.2024.107557

M3 - Article

C2 - 39002668

SN - 0021-9258

VL - 300

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 8

M1 - 107557

ER -