ABSTRACT. There is now convincing evidence that the Pig-a gene is mutated in patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease in which one or more clones of hematopoietic cells have incomplete assembly of glycosylphosphatidylinositol (GPI) anchors and absence of GPI-linked protein expression on the cell surface. Little is known, however, about the Pig-a protein product that is necessary for GPI anchor bioassembly. Relatively few substitution (missense) Pig-a gene mutations have been identified, but we noted two apparent clusters at codons 128-129 and 151-156 and hypothesized that these might represent critical regions of the Pig-a protein. We therefore used site-directed mutagenesis to create conservative mutations in the Pig-a protein, then performed structural and functional analysis. Each Pig-a mutation generated a Pig-a protein of normal size and stability, but certain mutations had substantial deleterious effects on protein function. Conservative mutation of codons histidine 128 (H128), serine 129 (S129), and serine 155 (S155) had greatly diminished function, while mutations of flanking residues had no effect on function. Our results represent the first structure/function analysis of the Pig-a protein, and suggest that codons H128, S129, and S155 represent critical regions of the Pig-a protein. Our results also suggest a means by which transgenic mice with a "partial knock-out" of Pig-a function could be generated, which would allow investigation of PNH in an animal model.

Keywords: Paroxysmal nocturnal hemoglobinuria, mutagenesis, GPI-linked proteins, protein function.

Reprint requests to: Russell E. Ware, M.D.,Ph.D., P.O. Box 2916, Duke University Medical Center, Durham, NC 27710, phone: (919) 684-5665, fax: (919) 684-5752, ware0005@mc.duke.edu.

ABSTRACT. Regulation of myeloid cell proliferation and differentiation in the bone marrow is mediated, in part, by the interaction of integrins on early myeloid cells with the extracellular matrix proteins secreted by stromal cells. To further define adhesive protein receptors of early myeloid cells, we examined the expression of the integrin GPIIb-IIIa (alphaIIbbeta3) in leukemic cell lines KG-1a, KG-1, and HL-60, that represent early stages of myeloid differentiation. All three cell lines expressed surface GPIIb-IIIa as measured by flow cytometry and by binding of 125I-anti-GPIIb-IIIa monoclonal antibody. Preincubation of cells with human AB serum or platelet releasate increased GPIIb-IIIa surface expression. GPIIb transcripts were identified in all three cell lines by Northern blot analysis. Furthermore, we readily detected GPIIb transcripts in fluorescence activated cell sorted (FACS) myeloid cells from normal human bone marrow by RT-PCR. Cloning and sequencing of the PCR products established the identity of GPIIb transcripts in the leukemic cell lines and CD34+/CD33+ normal bone marrow cells. Since the normal myeloid cells also demonstrated markers corresponding to the maturational stage of KG-1a/KG-1 cells, we propose that GPIIb-IIIa may serve as a myeloid differentiation antigen and as a key integrin of myeloid precursors.

Keywords: Integrins, glycoprotein IIb-IIIa, myeloid precursor cell, myeloid differentiation antigen, myeloid leukemic cell lines.

Reprint requests to: Lisa K. Jennings, Ph.D., Department of Medicine, Room A303, 956 Court Avenue, Memphis, Tennessee 38163, phone: (901) 448-5067, fax: (901) 448-7181, e-mail: ljennings@utmem1.utmem.edu.

ABSTRACT. A polymorphic site has been found in the human glucose phosphate isomerase (GPI) gene. This site is produced by a A G substitution at nt 489 of GPI cDNA, resulting in a silent mutation. To our knowledge, it is the first defined polymorphism in the gene. It is present with similar gene frequencies in Asian, American White, African American, and Jewish populations.

Keywords: Asian, Jewish, White American, restriction site.

Reprint requests to: Ernest Beutler, M.D., Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, phone: (619) 784-8040, fax: (619) 784-2083, e-mail: beutler@scripps.edu.