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G M Prince

Publications and source records attributed to G M Prince.

16 recordsLinked to original sources
PubMed2000

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Antigens, CD34↗
PubMed1996

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

Acyltransferases↗
PubMed1995

Peripheral blood harvest of unaffected CD34+ CD38- hematopoietic precursors in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) arises from somatic mutation of a bone marrow progenitor that disrupts glycosylinositol phospholipid (GPI) anchoring of cell surface proteins. We recently characterized the expression of GPI-anchored decay acclerating factor (DAF) and CD59 during hematopoietic development in PNH marrow. We found that, although a subset of early hematopoietic precursors identified by the CD34+CD38- phenotype exhibits normal DAF and CD59 expression, DAF and CD59 are absent on the majority of CD34+CD38- cells. Pluripotent CD34+CD38- hematopoietic stem cells normally circulate in the peripheral blood and can be collected by apheresis, cryopreserved, and later used for reconstitution of hematopoiesis. In this study, we examined the phenotypes of CD34+ cells that are released into the blood of PNH patients. Analyses of apheresis samples from three affected individuals showed discrete populations of circulating DAF+CD59+CD34+ and DAF-CD59-CD34+ cells. Variable proportions of CD34+CD38- cells were present within the peripheral blood CD34+ cells of each patient, but in all three cases the DAF+CD59+CD34+CD38- cell subset subset. Because CD34+ cells lacking CD38 antigen are highly enriched for self-renewing hematopoietic stem cells, these findings indicate that apheresis samples can serve as a source of unaffected stem cells for autologous marrow transplantation of PNH patients.

ADP-ribosyl Cyclase↗
PubMed1993

A candidate mammalian glycoinositol phospholipid precursor containing three phosphoethanolamines.

The glycoinositol phospholipid (GPI) anchors of mammalian proteins contain linear ethanolamine (EthN)-P-6ManManManGlcN glycan sequences that bear additional EthN-P substituents and in some cases include a fourth Man and a GalNAc or sialic acid-GalGalNAc. Precursors of these anchoring structures are preassembled in the endoplasmic reticulum by sequential glycosylation of inositol phospholipid. In previous studies (Hirose, S., Prince, G. M, Sevlever, D., Ravi, L., Rosenberry, T. L., Ueda, E., and Medof, M. E. (1992) J. Biol. Chem. 267, 16968-16974), a series of putative intermediates of this assembly process were isolated from human HeLa cells and murine lymphomas, and several of the more polar products were found to contain a second EthN-P attached to the Man residue (Man 1) proximal to GlcN. In this study, the most polar HeLa cell GPI species was purified by normal phase HeLa cell GPI species was purified by normal phase Iatrobead high performance liquid chromatography, and its glycan was characterized. Dionex anion exchange chromatographic analyses of fragments produced by nitrous acid deamination, hydrofluoric acid dephosphorylation, and trifluoroacetic acid hydrolysis in conjunction with biosynthetic labeling studies indicated a structure containing a third EthN-P substituent linked to the 6-position of Man 2. The polar GPI product exhibited a ManManManGlcN core lacking additional Man or GalNAc. The implications of the identification of this triply phosphoethanolamine-substituted species to mammalian GPI anchor biosynthesis are discussed.

Carbohydrate Sequence↗
PubMed1992

Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells. Localization of phosphoethanolamine.

A number of mammalian cell surface proteins are anchored by glycoinositol phospholipid (GPI) structures that are preassembled and transferred to them in the endoplasmic reticulum. The GPIs in these proteins contain linear ethanolamine (EthN)-phosphate (P)-6ManManManGlcN core glycan sequences bearing an additional EthN-P attached to the Man residue (Man 1) proximal to GlcN. The biochemical precursors of mammalian GPI anchor structures are incompletely characterized. In this study, putative [3H]Man-labeled GPI precursors were obtained by in vitro GDP-[3H] Man labeling of HeLa cell microsomes and by in vivo [3H]Man labeling of class B and F Thy-1 negative murine lymphoma mutants known to accumulate incomplete GPIs. The high performance liquid chromatography-purified in vitro and accumulated in vivo GPI products were structurally analyzed by nitrous acid deamination, hydrofluoric acid, trifluoroacetic acid hydrolysis, biosynthetic labeling, and exoglycosidase treatment. The data were consistent with a biosynthetic scheme in which Man and EthN-P are added stepwise to the developing glycan. Several additional points were demonstrated: 1) putative mammalian GPI precursors contain incomplete core glycans corresponding to those in previously characterized trypanosome GPI precursors. 2) The proximal EthN-P found in mature mammalian GPI anchor structures is added to Man 1 prior to incorporation of Man 2 and Man 3. 3) Glycans in the incomplete GPIs that accumulate in classes B and F lymphoma mutants consist of Man2- and Man3GlcN in which EthN-P is linked to Man 1. 4) Distal EthN-P linked to the 6-position of Man, characteristic of the complete GPI core, is found both in a subsequent GPI species with the glycan sequence EthN-P-6ManMan(EthN-P----)ManGlcN and in a more polar GPI product.

Animals↗
PubMed1992

Synthesis of mannosylglucosaminylinositol phospholipids in normal but not paroxysmal nocturnal hemoglobinuria cells.

To identify mannosyl (Man)-containing intermediates of the human glycoinositol phospholipid (GPI) anchor pathway and examine their expression in paroxysmal nocturnal hemoglobinuria (PNH), mannolipid products deriving from in vitro guanosine diphosphate [3H]Man labeling of HeLa cell microsomes were characterized. The defined GPI species were correlated with products deriving from in vivo [3H]Man labeling of normal and (GPI-anchor defective) affected leukocytes. In vitro analyses in HeLa cells showed dolichol-phosphoryl (Dol-P)-[3H]Man and a spectrum of [3H]Man lipids exhibiting TLC mobilities approximating those of Trypanosoma brucei (Tryp) GPI precursors. Iatrobead HPLC separations and partial characterizations of the major isolated [3H]Man species (designated H1-H8) showed that all but H1 (Dol-P-Man) were sensitive to HNO2 deamination and serum GPI-specific phospholipase D digestion but were resistant to phosphatidylinositol-specific phospholipase C digestion unless previously deacylated with mild alkali. [3H]Man label in H3, H4, and H6 but not in H5 or H7 was efficiently released into the aqueous phase by jack bean alpha-mannosidase digestion. BioGel P-4 and AX-5 sizing of the dephosphorylated core glycan fragments of H6 and H7 gave values that coincided precisely with the corresponding glycan fragments from the fully assembled Tryp anchor donor A' (P2). Affected leukocytes from four patients with PNH supported formation of GlcNAc- and GlcN-PI but all failed to express H6 and H7 as well as H8 and two showed complete absence of earlier Man-containing intermediates. These findings argue that human intracellular GPI mannolipids are built on acylated inositol phospholipids, that H6 and H7 contain differentially phosphoethanolamine-substituted Man3-GlcN-inositol cores, and that PNH cells are defective in conversion of GlcN-PI into these more mature mannolipid structures.

Antigens, CD↗
PubMed1992

Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59.

CD59, the membrane regulator of autologous C5b-9 channel formation, exhibits variable sensitivity to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that releases glyco-inositolphospholipid (GPI)-anchored proteins from cell surfaces. To determine whether the GPI-anchor phospholipid of CD59 is similar to that of decay-accelerating factor (DAF) and whether variation in its structure underlies its variable enzyme susceptibility, the GPI anchors of the two proteins expressed on erythrocytes, polymorphonuclear and mononuclear leucocytes were compared in situ and after purification. Flow cytometric analyses of PI-PLC-treated cells showed parallel cell type specific release of both proteins as a function of enzyme concentration. Non-denaturing PAGE analyses of alkaline/hydroxylamine-treated proteins (affinity-purified from [125I]-surface-labelled cells) provided evidence for (i) comparable proportions of GPI-anchor acylation, and (ii) alkali-resistant rather than alkali-sensitive lipid substituents in erythrocytes. These findings argue that the differential C5b-9 sensitivity that distinguishes paroxysmal nocturnal haemoglobinuria II and III erythrocytes does not derive from expression of CD59 molecules with alternative GPI-anchor phospholipid structures.

Antigens, CD↗
PubMed1985

Factor I co-factor activity of CR1 overcomes the protective effect of IgG on covalently bound C3b residues.

We have shown previously that C3b resides in a protected site when it is covalently bound to IgG (C3b-IgG). Such C3b displays a reduced affinity for factor H, with consequent enhanced survival in the presence of factors H and I and increased capacity for promoting alternative pathway consumption of C3. Because erythrocyte CR1 may be a major co-factor for factor I-mediated inactivation of immune complex-borne C3b in blood, we have examined the effect of covalently bound IgG on the C3b-CR1 interaction. Binding of monomeric C3b and C3b-IgG to human erythrocyte CR1 demonstrates identical ionic strength dependence for both species. Identical numbers of binding sites with indistinguishable affinities are detected by both ligands. Cleavage of the alpha'-chain of C3b and the alpha'-heavy chain of C3b-IgG proceeds at the same rate when erythrocyte CR1 serves as co-factor for factor I. Unlike factor H, CR1 supports a second cleavage of fluid-phase iC3b alpha'1 chain (free or bound to IgG) that generates C3c and a 33,000 m.w. fragment, which bears antigenic markers characteristic of C3g. Inactivation of C3b and C3b-IgG by CR1 and factor I also occurs at physiologic ionic strength, but proceeds very slowly relative to rates attainable with sub-physiologic inputs of factor H. CR1 does not recognize IgG-bound C3b as being in a protected site but, because of low binding affinity at physiologic ionic strength, is probably highly dependent on multivalent ligand-receptor interactions to efficiently exert its co-factor functions. Thus, inactivation of C3b-IgG heterodimers or small immune complexes bearing limited numbers of C3b residues may remain largely factor H-dependent in vivo, with resultant enhanced C3b survival.

Antigen-Antibody Complex↗
PubMed1983

Requirement for human red blood cells in inactivation of C3b in immune complexes and enhancement of binding to spleen cells.

We showed previously that soluble immune complexes (IC) added to normal human serum and unseparated autologous blood cells bind predominantly to red blood cells (RBC) bearing C3b receptors. RBC-associated IC are processed both by C3b/C4b inactivator (I) cleavage of C3b and by alternative pathway-dependent C3b generation, and are returned to the serum in a modified form. In the present study, soluble IC were prepared by using 125I-BSA and guinea pig anti-BSA, and were treated with diluted human serum to optimize sustained C3b-dependent binding to human RBC. This binding to RBC could not be abrogated by preincubation of IC with heat-inactivated human serum or purified serum regulatory proteins, I and beta 1H (H), but after binding, pretreated IC dissociated. When IC were prepared with serum containing 125I-C3, treatment with I + H did not release labeled C3 from the complexes. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of 125I-C3b in IC showed similar labeled peaks whether IC were pretreated with buffer or with I + H or were bound to RBC without pretreatment. In contrast, when IC containing 125I-C3b were exposed to I + RBC, nearly 70% of the label was released from the IC. The released material analyzed by SDS-PAGE showed major 125I-C3 peaks corresponding in m.w. to free beta-chain and alpha 1 cleavage products of 30,000 to 33,000 daltons. IC in undiluted serum after incubation with RBC gave less binding to human polymorphonuclear leukocytes than after incubation without RBC, but notably more binding to guinea pig spleen cells and human lymphoblastoid cells. These findings argue that exposure to serum regulatory proteins does not prevent subsequent binding of experimental IC to RBC, that RBC binding promotes degradation of C3b in IC which proceeds beyond iC3b, and that this I-mediated breakdown renders IC reactive with spleen cells.

Animals↗
PubMed1983

Immune complex alterations occur on the human red blood cell membrane.

Experimental antigen-antibody complexes (Ag-Ab) were incubated at 37 degrees with human red blood cells (RBC) suspended in autologous normal serum and the reaction stopped after progressively increasing times. Bound antigen-antibody-complement complexes (Ag-Ab-C) were eluted from C3b receptors and the eluted Ag-Ab-C re-incubated with different blood cell types suspended in serum, or centrifuged (along with unbound Ag-Ab-C found in the serum) through 20-50% sucrose gradients. Ag-Ab-C recovered from C3b receptors shortly after initial binding to RBC bound efficiently to other RBC, polymorphonuclear and mononuclear cells, and sedimented rapidly. Ag-Ab-C simultaneously present in the serum sedimented with a similar velocity. Ag-Ab-C recovered at a subsequent time during RBC interaction bound less well to each blood cell type, and sedimented less rapidly. Decreased amounts of rapidly sedimenting Ag-Ab-C were present in the serum. Ag-Ab-C recovered from C3b receptors at a still later time in the course of RBC interaction bound poorly to each cell type, and sedimented slowly. Increased amounts of slowly sedimenting Ag-Ab-C were found in the serum. These findings indicate that alterations in properties of immune complexes can occur while they are associated with C3b receptors on RBC membrane in solid phase.

Antigen-Antibody Complex↗
PubMed1983

Alternative pathway-mediated rebinding of immune complexes to human red blood cells.

Antigen-antibody complexes (Ag-Ab) prepared from 125I-bovine serum albumin (BSA) and guinea-pig anti-BSA were (i) incubated at 37 degrees for 4 min with undiluted normal human serum and autologous red blood cells (RBC) together, or (ii) incubated first at 37 degrees for 30 min with serum diluted optimally for binding and then with RBC. Reactions were stopped by dilution and cooling, and RBC bearing antigen-antibody-complement complexes (Ag-Ab-C) were washed and resuspended in either untreated normal human serum (undiluted or diluted), serum treated with zymosan (SZYM), ethylenediamine tetracetic acid (SEDTA) or ethyleneglycol tetracetic acid-Mg++ (SEGTA), serum heated at 56 degrees for 30 or 120 min (S delta 30 or S delta 120), or buffer alone. The mixtures were placed at 37 degrees and the percentage of Ag-Ab-C dissociated from RBC after progressively increasing times determined. (i) Ag:Ab:C bound to RBC with undiluted serum dissociated more rapidly following resuspension in SZYM, SEDTA, or S delta 30 than following resuspension in untreated serum. Rate of dissociation in SEGTA paralleled that in untreated serum. (ii) Ag-Ab-C bound to RBC with diluted serum dissociated following resuspension in SZYM, SEDTA, or S delta 30, but rebound and dissociated a second time following resuspension in untreated serum or SEGTA. Initial dissociation occurred in less than 1 min in undiluted serum, took place at 0 degrees as well as 37 degrees, and was diminished but detectable in greater than 8- and greater than 64-fold-diluted serum, respectively. Rebinding required 37 degrees, factors B and D and C3, and was maximal at 4-8 min. Subsequent dissociation had similar complement requirements to initial dissociation, but occurred only at 37 degrees and was 90% complete at 15 min. No dissociation of Ag-Ab-C bound to RBC in (i) or in (ii) occurred following resuspension in S delta 120 or in buffer. These findings suggest that after initial binding, release of experimental immune complexes from RBC in whole serum involves concurrent dissociation and alternative pathway-dependent rebinding.

Antigen-Antibody Complex↗
PubMed1982

Release of soluble immune complexes from immune adherence receptors on human erythrocytes is mediated by C3b inactivator independently of Beta 1H and is accompanied by generation of C3c.

Antigen.antibody complexes (Ag.Ab) prepared from (125)I-labeled bovine serum albumin and guinea pig anti-albumin were incubated at 37 degrees C for 30 min with normal human serum diluted optimally for binding (1:16) and then with autologous erythrocytes (RBC). After washing, RBC-bearing antigen.antibody.complement complexes (Ag.Ab.C) were resuspended in serum reagents or solutions of purified complement components, and the kinetics of dissociation were analyzed. Ag.Ab.C dissociated in serum heated at 56 degrees C for 30 min (SDelta30) but not in serum heated for 120 min (SDelta120). Dissociation in SDelta30 markedly decreased after adsorption with anti-C3b inactivator but not anti-beta1H or anti-C4 binding protein (C4bp), and dissociation in SDelta120 markedly increased after addition of C3b inactivator. Hemolytic assays revealed that SDelta30 retained inactivator activity whereas SDelta120 lacked significant activity. Ag.Ab.C dissociated in the presence of purified inactivator or C3b but not beta1H or C3. Dissociation was more rapid with inactivator than with C3b and occurred at 0 degrees C as well as at 37 degrees C. Treatment with inactivator inhibitor abolished dissociation in SDelta30; dissociation in inactivator deficient serum was markedly reduced. Addition of beta1H did not enhance inactivator-mediated dissociation at limiting dilutions of inactivator, and adsorption of Ag.Ab.C with anti-beta1H or preparation of Ag.Ab.C with serum adsorbed with anti-beta1H did not diminish dissociation. After dissociation with inactivator, Ag.Ab.C were unchanged in size but were no longer able to bind to fresh RBC and gave enhanced binding to Raji and Daudi lymphoblastoid cells. NaDodSO(4)/polyacrylamide gel electrophoresis of Ag.Ab.C prepared with (125)I-labeled C3 revealed that, after binding to RBC, dissociation with inactivator was accompanied by generation of a C3 fragment the size of C3c. Preincubation of Ag.Ab.C with excess inactivator did not prevent subsequent binding of Ag.Ab.C to RBC but, immediately after binding, Ag.Ab.C dissociated rapidly. These findings indicate that C3b inactivator can release immune complexes from immune adherence receptors on human RBC, that release occurs independently of beta1H, alters cell binding properties of immune complexes, and involves multiple cleavages of the C3b alpha' chain, and that receptors in human RBC membrane are required for this C3b inactivator-mediated breakdown.

Antigen-Antibody Complex↗
PubMed1982

Restoration of normal interactions between immune complexes and systemic lupus erythematosus blood cells by normal serum.

Blood cells from patients with systemic lupus erythematosus (SLE) and from healthy control subjects were studied for their ability to bind exogenous immune complexes. Cells were mixed with experimental complexes (prepared from 125I-radiolabeled bovine serum albumin [BSA] and guinea pig antiserum to BSA) after incubation of the complexes with autologous serum in variable proportions. Unfractionated blood cells from SLE patients bound fewer added complexes (mean +/- SD = 39.8% +/- 12.9%) than cells from normal controls (mean +/- SD = 63.6% +/- 9.5%) (p less than 0.001); as much as 128-fold (mean = 23-fold) higher (p less than 0.01) proportions of serum to complex were required to optimize the lower binding. When SLE blood cells were mixed with complexes preincubated with normal sera instead of autologous SLE sera, SLE cells bound increased amounts of added complexes, and binding was optimal at the same lower proportions of serum to complex as for normal cells. When normal cells were mixed with complexes preincubated with compatible SLE sera in place of endogenous normal sera, binding to normal cells decreased to SLE levels, and the same higher proportions of serum to complex were required to optimize the lower binding as for the SLE cells. Similar results were obtained when binding to purified mononuclear cell preparations from SLE patients and controls was studied. When comparative studies were performed at high complex-to-cell ratios with complexes prepared using normal sera, SLE cells bound fewer complexes per cell than normal cells. These results indicate that although abnormalities in SLE cells are demonstrable, the major factor accounting for aberrant interaction between immune complexes and SLE cells resides in alterations in SLE sera.

Adolescent↗
PubMed1982

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.

Antigen-Antibody Complex↗