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. 2019 Sep 30;13(3):324-333.
eCollection 2019 Sep.

Characterization of Glycoproteins of Native 19kDa C-Terminal Merozoite Surface Protein-1 from Native Antigen of Plasmodium falciparum

Sahar Tajik  1 , Sedigheh Sadeghi  2 , Ayda Iravani  2 , Mitra Khalili  1 , Mohammad Arjmand  2 , Nassir-Ud Din  3 , Farideh Vahabi  2 , Hossein Feiz-Haddad  4 , Behzad Lame-Rad  1 , Saied Reza Naddaf  5 , Zahra Zamani  2
Affiliations

Characterization of Glycoproteins of Native 19kDa C-Terminal Merozoite Surface Protein-1 from Native Antigen of Plasmodium falciparum

Sahar Tajik et al. J Arthropod Borne Dis. .

Abstract

Background: Plasmodium falciparum is the protozoan parasite which causes malignant malaria of medical concern. Prime candidates for recombinant vaccine development are asexual stage antigens of P. falciparum, for example, merozoite surface proteins (MSP1 and MSP2) not given satisfactory results to date. In this study, the 19kDa C-terminal of MSP1, a vaccine candidate was purified in its native form in the ring stage, and its glycoproteins studied.

Methods: The study was carried out at the Biochemistry Department of Pasteur Institute of Iran in the years 2015-2016. Large scale culture of P. falciparum was performed in vitro with 80% ring stage parasitemia. Isopycnic ultracentrifugation with 36% sucrose and analytical SDS-PAGE on the supernatant and precipitate performed, and the 19kDa antigen was obtained by cutting it from strips of preparative SDS gels. Purified protein was concentrated and analyzed by SDS-PAGE and immunoblotting, using antibodies raised to recombinant C-terminal MSP1.

Results: The purified protein gave a single band of 19kDa antigen as shown by silver staining of SDS-PAGE and a single bond in immunoblotting. Bioinformatics also confirmed the likelihood of the presence of glycans on the antigen.

Conclusion: The presence of N and O-glycoproteins were detected by Q proteome kit. This work was done on the ring stage, and earlier workers confirmed the presence of glycoproteins on MSP1 in the other stages. This glycosylation is present in all stages, and maybe incomplete protection elicited by recombinant MSP1 antigens is due to lack of N and O-glycoproteins.

Keywords: C-terminal 19kDa; Glycoproteins; Merozoite surface protein1; Plasmodium falciparum.

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Figures

Fig. 1.
Fig. 1.
Synchronized large scale preparation of ring-stage P. falciparum
Fig. 2.
Fig. 2.
Coomassie blue staining of SDS-PAGE of parasite extract. MW is the molecular weight marker, and the right lane is P. falciparum “parasite extract”
Fig. 3.
Fig. 3.
Preparative negative imidazole staining of SDS-PAGE of P. falciparum “parasite extract”. MW is molecular weight marker
Fig. 4.
Fig. 4.
Glycoprotein Schiff's staining of SDS PAGE of P. falciparum “parasite extract”. MW is molecular weight markers
Fig. 5.
Fig. 5.
Silver-staining of SDS-PAGE of P. falciparum of purified C-19 kDa antigen. MW is molecular weight markers
Fig. 6.
Fig. 6.
Immunoblotting of purified C-terminal 19kDa antigen using anti-C-terminal monoclonal antibody raised in rabbits. MW is molecular weight markers
Fig. 7.
Fig. 7.
Silver staining of SDS-PAGE of purified antigen from O-proteome kit. MW is molecular weight markers. Lane A purified antigen C-terminal 19kDa, lane B Glycoproteins containing galactose and galactosamine eluted from PNA Kit, lane C containing N-acetyl neuraminic, galactose and galactosamine eluted from AIL column
Fig. 8.
Fig. 8.
Silver staining of SDS-PAGE of purified antigen from total Q proteome kit, MW is molecular weight markers. Lane A purified antigen C-terminal 19kDa, lane B glycoproteins containing N-acetylglucosamine and sialic acid eluted from WGA column; lane C Mannose-containing glycoproteins eluted from Con A columns; lane D N-acetyl glucosamine and sialic acid glycoproteins eluted from WGA column
Fig. 9.
Fig. 9.
EXPASY file graphs showing 62 probable O-glycosylation sites of P. falciparum C-19kDa C-terminal MSP1 antigen and 5 potential N-glycan sites on the 250 base sequence
Fig. 10.
Fig. 10.
EXPASY file graphs showed five probable N-glycosylation sites of P. falciparum C-19kDa C-terminal MSP1 antigen
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