Molecular and Biochemical Parasitology 85 (1997) 125–129

Short communication

An additional primary proteolytic processing site in merozoitesurface protein-1 of Plasmodium berghei

Mark F. Wiser*, Carole S. Toebe1, Gregory J. Jennings2

Department of Tropical Medicine, Tulane Uni6ersity School of Public Health and Tropical Medicine, 1501 Canal Street,New Orleans, LA 70112, USA

Received 10 September 1996; revised 26 November 1996; accepted 29 November 1996

Keywords: Plasmodium berghei ; Malaria; Merozoite surface; Proteolytic processing; Vaccine

The merozoite surface protein-1 (MSP-1) is arelatively abundant protein which has been foundin all Plasmodium species examined [1,2]. MSP-1is synthesized as a high molecular mass (�200kDa) precursor protein which is proteolyticallyprocessed into smaller fragments. Proteolytic pro-cessing of MSP-1 is a two-step procedure charac-terized by primary and secondary processingevents [3]. Primary processing occurs at, or justbefore, terminal merozoite differentiation and re-lease and results in the formation of a noncova-lent polypeptide complex with fragments ofapproximately 83, 30, 38 and 42 kDa [4]. The 42

kDa C-terminal fragment is further processed to33 and 19 kDa fragments at the time of merozoiteinvasion [5]. This secondary processing is cata-lyzed by a Ca2+-activated serine protease whichresults in the shedding of a soluble MSP-1 com-plex [6]. The remaining glycosyl-phosphatidylinos-itol-anchored 19 kDa fragment [7] contains twoepidermal growth factor-like modules [8] and iscarried into the erythrocyte upon merozoite inva-sion [9].

Comparison of the MSP-1 sequence from P.berghei with other rodent parasite MSP-1 se-quences [10–12] revealed four major interspeciesvariable regions, or blocks (Jennings et al., inpreparation). The first (i.e., most N-terminal) ofthese variable blocks was cloned from a lgt11cDNA expression library screened with two dif-ferent mAbs against PbMSP-1 [13]. The mAbs,designated as mAb-F4.4 and mAb-L1.6, weregenerated from mice immune to P. berghei [14].Variable block 1 (VB1) contains seven degenerate

Abbre6iations: LT-B, B subunit of E. coli labile toxin; MSP-1, merozoite surface protein-1; VB1, variable block 1.

* Corresponding author. Tel.: +1 504 5842507; fax: +1504 5996686; e-mail: wiser@mailhost.tcs.tulane.edu

1 Present address: Department of Biology, City College ofSan Francisco, San Francisco, CA 94112, USA.

2 Present address: Michigan Department of CommunityHealth, P.O. Box 30035, Lansing, MI 48909, USA.

0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.

PII S 0 1 66 -6851 (96 )02811 -3

M.F. Wiser et al. / Molecular and Biochemical Parasitology 85 (1997) 125–129126

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M.F. Wiser et al. / Molecular and Biochemical Parasitology 85 (1997) 125–129 127

tandem repeats of 10 amino acids. A series ofrecombinant constructs, possessing or lacking thetandem repeats, were prepared by taking advan-tage of a SpeI restriction site (Fig. 1a), and weretested as vaccines (C.S. Toebe, Ph.D. Disserta-tion, Tulane University). Immunoblotting of theproteins expressed by these recombinant con-structs indicates that the epitope recognized bymAb-L1.6 is to the N-terminal side of the SpeIrestriction site (Fig. 1b), and the epitope recog-nized by mAb-F4.4 is the C-terminal side of theSpeI restriction site (Fig. 1d).

Analysis of schizont-infected erythrocytes andenriched merozoites by SDS gel electrophoresisand immunoblotting reveals that mAb-L1.6 rec-ognizes an approximately 30 kDa polypeptide notrecognized by mAb-F4.4 (Fig. 1c), and mAb-F4.4recognizes an approximately 60 kDa polypeptidenot recognized by mAb-L1.6 (Fig. 1e). BothmAb-L1.6 and mAb-F4.4 recognize the intact 230kDa MSP-1 and an approximately 90 kDapolypeptide (Fig. 1c,e). This 90 kDa polypeptideis homologous to the 83 kDa processing fragmentof P. falciparum MSP-1 as demonstrated by align-ment of the rodent Plasmodium MSP-1 sequenceswith PfMSP-1 sequences [13]. The immunoblot-ting results indicate that the 90 kDa PbMSP-1fragment is further processed into 30 kDa and 60kDa polypeptides and the protease site is betweenthe L1.6 and F4.4 epitopes. A further processingof the 83 kDa fragment to 73 or 67 kDa frag-ments has been occasionally reported for PfMSP-1 [15,16]. This further processing of the 83 kDafragment, however, appears to be a minor event inthat the 83 kDa polypeptide is usually the pre-dominant processing product. Therefore MSP-1from P. berghei has a proteolytic processing sitethat is absent in PfMSP-1.

O’Dea et al. [17] have also proposed an addi-tional protease processing site in the N-terminalregion of MSP-1 from P. chabaudi which givesrise to 35 and 52 kDa fragments. N-terminalsequencing of the 52 kDa fragment from PcMSP-1 reveals that cleavage occurs between an as-paragine and a threonine residue (denoted byunderlining in Fig. 1a). Alignment of the MSP-1sequences from other rodent parasites reveals that

Table 1Distribution of amino acids in the primary processing sites ofMSP-1 from rodent Plasmodium species

P2% P3%P6 P5 P4 P3 P2 P1 P4%P1%

12E 6E6I 4V 6A 4S 10G 8E 5E15S2S 3A4Q 5D4I 1T5T 4T 6A 6Q2T 2Q3P 3T 3V 3T3R 1N

2D2T2T 2A 1I 2E 1S1N1H 2V1Q 1S 2N

1G1R 1G1K

The four rodent Plasmodium sequences were aligned withregard to the four primary processing sites of P. chabaudi [17].Accession numbers for the sequences are listed in the legend toFig. 1. The number of occurrences of particular amino acids(single-letter code) found at positions surrounding the 16 (fourstrains×four primary processing sites) scissile bonds (betweenP1 and P1% according to the nomenclature of Schechter andBerger [22]) are shown.

the scissile bond is on the boundary between aconserved region and VB1 (Fig. 1a) and that thescissile bonds differ between species and strains.However, the homologous residues in P. bergheiand P. yoelii, glutamine and serine, are chemicallysimilar to asparagine and threonine. A mAbagainst PyMSP-1 immunoprecipitates polypep-tides of 230, 90 and 56 kDa and pulse-chaseexperiments demonstrate that the 230 kDa proteinis converted to the 90 and 56 kDa fragments [18].The similarity in the sizes of the P. berghei and P.yoelii MSP-1 fragments (i.e. 60 vs. 56 kDa) sug-gests that this additional proteolytic processingalso occurs in P. yoelii. No information is avail-able on the processing of MSP-1 from the IP-PC1strain of P. chabaudi.

The amino acids flanking all four primary pro-cessing sites [17] in the MSP-1 sequences fromfour rodent parasites were analyzed (Table 1).The amino acids surrounding the scissile bondsare somewhat conserved and have a strong predis-position for certain types of amino acids. Polarand/or charged amino acids are found in the P1and P3 positions, whereas the P2 and P4-P6 posi-tions tend to be aliphatic. On the P% side of thescissile bond the amino acids tend to have hy-droxyl or acidic side groups giving rise to some-what negatively charged domain. We propose a

M.F. Wiser et al. / Molecular and Biochemical Parasitology 85 (1997) 125–129128

consensus sequence of h-h-h-p-G/A-E/Q¡S-E-n-n, where the three lower case h’s refer to astretch of generally non-polar amino acids inwhich at least one is hydrophobic, the lowercase p refers to a polar amino acid and the twolower case n’s refer to a tendency to be nega-tively charged. The primary processing sites ofPfMSP-1 [17] also exhibit this same general con-sensus sequence, except that the most C-terminalprimary processing sites of PfMSP-1 lacks thestrong negative tendency on the P% side of theproposed consensus sequence. However, alterna-tive processing sites for this position, which per-fectly match the consensus sequence, have beenproposed [19]. In addition, some minor process-ing fragments have been sequenced [16] andthese protease sites also show good, but imper-fect, homology to the consensus sequence.Cooper and Bujard [20] have described aschizont membrane associated protease whichcan cleave recombinant MSP-1. The sequencesrecognized by this protease all comply with theproposed consensus sequence.

In summary, the collective data indicate thatMSP-1 from rodent Plasmodium species has anadditional primary processing site that is likelyabsent in P. falciparum. The evidence includes:the similar sizes of the MSP-1 proteolytic frag-ments (30–35 kDa+52–60 kDa), the strongamino acid homology in this region (Fig. 1a),and the conservation of the sequence flankingthe scissile bond as compared to the other pri-mary processing sites (Table 1). Furthermore,the similarity in the sequences of the primaryprocessing sites suggest that a single protease isresponsible for the primary processing. In addi-tion, since they recognize different processingfragments, mAb-L1.6 and mAb-F4.4 will be use-ful reagents for the study of MSP-1 processing.

Acknowledgements

We thank Dr John Lonsdale-Eccles (Univer-sity of Alabama, Birmingham) for helpful dis-cussions on proteases and his critical commentson the manuscript.

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