Molecular Cloning and Characterization of a Novel Repeat-containing Leishmania major Gene,ppg1, That Encodes a Membrane-associated Form of Proteophosphoglycan with a Putative Glycosylphosphatidylinositol Anchor*
- From the ‡Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-72076 Tübingen, Germany and the ¶Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, 3050 Victoria, Australia
Abstract
Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene,ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(±S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) – .
-
↵§ To whom correspondence should be addressed. Fax: 49–7071-601235; E-mail: thomas.ilg@tuebingen.mpg.de.
-
↵2 E. Handman, unpublished results.
-
↵3 T. Ilg and E. Handman, unpublished results.
-
↵4 T. Ilg, unpublished results.
- Abbreviations:
- LPG
-
lipophosphoglycan
- PPG
-
proteophosphoglycan
- fPPG
-
filamentous PPG
- mPPG
-
membrane-bound PPG
- GPI
-
glycosylphosphatidylinositol
- ELISA
-
enzyme-linked immunosorbent assay
- DIG
-
digoxygenin
- PBS
-
phosphate-buffered saline
- TBS
-
Tris-HCl-buffered saline
- TBS/T
-
TBS containing Tween 20
- TBS/MT
-
TBS containing Tween 20 and skim milk powder
- PCR
-
polymerase chain reaction
- PAGE
-
polyacrylamide gel electrophoresis
- BSA
-
bovine serum albumin
- contig
-
group of overlapping clones
- mAb
-
monoclonal antibody
- PVDF
-
polyvinylidene difluoride
- bp
-
base pair(s)
- kb
-
kilobase pair(s)
- ORF
-
open reading frame
- RT
-
reverse transcription
- SN
-
supernatant
- P
-
pellet
-
- Received April 21, 1999.
- Revision received July 14, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
