Proteophosphoglycans of Leishmania mexicana. Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes.
Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressed ppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.
Göpfert
U
U
Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-72076 Tübingen, Germany.
Goehring
N
N
Klein
C
C
Ilg
T
T
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018390:Gene Targeting; D015801:Helminth Proteins; D015151:Immunoblotting; D007894:Leishmania mexicana / Q000235:genetics*; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000737:chemistry / Q000235:genetics*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid
D015801:Helminth Proteins; C405069:Ppg2 protein, Leishmania mexicana; D011509:Proteoglycans; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012313:RNA