A NOVEL STRATEGY FOR THE SYNTHESIS OF THE CYSTEINE-RICH PROTECTIVE ANTIGEN OF THE MALARIA MEROZOITE SURFACE PROTEIN (MSP-1) - KNOWLEDGE-BASED STRATEGY FOR DISULFIDE FORMATION

The most promising antigen for a protective malaria vaccine is a cyste ine-rich domain at the carboxyr terminus of the merozoite surface prot ein (MSP-1). Passive transfer of anti-MSP-1 antibody or immunization o f MSP-1 against infection challenge confers protection in primate and rodent models. The antigen belongs to the three-disulfide epidermal gr owth factor (EGF) family based on the alignment of the six cysteines. In the K1 strain there are, however, only four cysteines corresponding to the four carboxyl cysteines of EGF. Furthermore, disulfide pairing would produce a non-EGF pattern. Because this cysteine-rich antigen i s conformation-dependent, and reduction of the disulfide bonds abolish es antigenicity, we used a synthetic analog to investigate the probabl e disulfide pairing of this antigen. This paper describes the synthesi s, Folding and disulfide pairings of two 50-residue cysteine-rich pept ides. One contains two disulfides (VK-50) derived from the native sequ ence of MsP-1 of the Thailand K1 strain (aa 1629-1679). The other cont ains an EGF-like, three-disulfide [Cys-9,14]VK-50 peptide. Both peptid es were synthesized by a solid-phase method using Fmoc-chemistry. The crude peptide of VK-50 was folded, and the disulfide was oxidized by t he DMSO method to obtain a structure with an expected disulfide pairin g of 3-4, and 5-6. The specific pairing pattern of 1-3, 2-4 and 5-6 in [Cys 9,14]VK-50 corresponding to EGF in [Cys 9,14]VK-50 was obtained using a 'knowledge-based' (KB) strategy for their formation. Purified VK-50 and [Cys-9,14]VK-50 had the correct molecular weight, as shown b y Cf-252 fission ionization mass-spectrometry. The disulfide pairings were confirmed by enzymatic digestion. These two peptides can be conju gated to the multiple peptide antigen core for immunization. The immun ological results will allow us to conclude the correct disulfide pairi ng and the conformational importance of this antigen. (C) Munksgaard 1 994.