The carboxyl terminus of Pneumocystis carinii glycoprotein A encodes a functional glycosylphosphatidylinositol signal sequence.
Pneumocystis carinii pneumonia is a hallmark disease associated with AIDS. An abundant glycoprotein, termed gpA, on the surface of P. carinii is considered an important factor in host-parasite interactions. The primary structure of ferret P. carinii gpA contains a carboxyl-terminal sequence characteristic of a signal for glycosylphosphatidylinositol (GPI) anchors. Here we report the capacity for this gpA carboxyl sequence to direct attachment of a secreted protein, human growth hormone (hGH), to the membranes of COS cells. A control fusion protein (hGHDAF37) was obtained which, under the direction of the GPI signal from decay accelerating factor, directs hGH cell surface expression. A construct (phGH2-1A30) was created similar to hGHDAF37 by fusing hGH to the putative GPI signal sequence encoded in the terminal 30 residues from a ferret P. carinii gpA cDNA clone. By indirect immunofluorescent staining, hGH was detected on the surface of COS cells transfected with phGH2-1A30; this surface location was confirmed by confocal laser cytometry. Metabolic labeling with [3H]ethanolamine and subsequent immunopurification of hGH from cells transfected with phGH2-1A30 confirmed that a lipid moiety characteristic of a conventional GPI anchor was linked covalently to hGH, and cell surface hGH2-1A30 fusion protein was sensitive to enzymatic cleavage by phosphatidylinositol-phospholipase C. Furthermore, hGH2-1A30 recombinant protein cofractionated with 5'-nucleotidase, a classical GPI-anchored membrane marker. Together, these results indicate that the carboxyl-terminal residues of ferret P. carinii gpA constitute a biologically functional GPI consensus domain, thus providing a potential mechanism for antigenic variation of P. carinii gpA during P. carinii pneumonia.
Guadiz
G
G
Departments, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Haidaris
C G
CG
Maine
G N
GN
Simpson-Haidaris
P J
PJ
D015720:5'-Nucleotidase / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D018958:CD55 Antigens / Q000378:metabolism; D019556:COS Cells; D019856:Ethanolamine / Q000378:metabolism; D005289:Ferrets; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000737:chemistry*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D019382:Human Growth Hormone / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011010:Pneumocystis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D010738:Type C Phospholipases / Q000378:metabolism
D018958:CD55 Antigens; D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; C077556:GPA protein, Pneumocystis carinii sp.; D019856:Ethanolamine; D015720:5'-Nucleotidase; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase