2 TANDEMLY ARRAYED GENES ENCODE THE (HISTIDINE) SECRETORY ACID-PHOSPHATASES OF LEISHMANIA-DONOVANI

Leishmania donovani promastigotes constitutively secrete a glycosylate d and phosphorylated acid phosphatase activity. This secretory acid ph osphatase (SAcP) was purified from L. donovani culture supernatants an d amino-acid sequence was obtained from both the N-terminus and a tryp tic peptide fragment derived from the isolated protein. A polymerase c hain reaction (PCR)-based strategy, using degenerate oligo primers des igned from the amino-acid sequence data, identified two single-copy, t andemly arrayed open reading frames (ORFs) capable of encoding the L. donovani SAcP (SAcP-1, 2052 bp and SAcP-2, 2124 bp). Both SAcP-1 and - 2 were shown to be actively transcribed by L. donovani promastigotes b y reverse transcription (RT) and PCR amplification. The deduced amino- acid sequences of SAcP-1 and SAcP-2 show high conservation to each oth er in four regions: a 23-amino-acid signal peptide; a catalytic domain containing several potential N-linked glycosylation sites; a Ser/Thr- rich repeat region containing multiple potential phosphorylation sites and a common C-terminus. Within the catalytic domain, the L. donovani SAcPs possess two conserved consensus sequences characteristic of his tidine acid phosphatases (AcPs). Furthermore, antisera to native L. do novani SAcP immunoprecipitated in vitro transcription/translation prod ucts of both SAcP-1 and SAcP-2. Cumulatively, these data indicate that the acid phosphatase activity constitutively secreted by L. donovani promastigotes is composed of two (histidine) AcP isoforms that are enc oded by SAcP-1 and SAcP-2, respectively. (C) 1997 Elsevier Science B.V .