Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants

Robert P. Mohney, Jansen J. Knez, Lakshmeswari Ravi, Daniel Sevlever, Terrone L. Rosenberry, Shinichi Hirose, M. Edward Medof

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Abstract

Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992). J. Biol. Chem. 267, 5272- 5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway- defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine- inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan (EthN-P→)ManGlcN- and EthN-P-6Man(EthN-P- 6)Man(EthN-P→)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D- glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.

Original languageEnglish (US)
Pages (from-to)6536-6542
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number9
StatePublished - Mar 4 1994
Externally publishedYes

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Anchors
Lymphoma
Phospholipids
Ethanolamine
Phosphatidylinositols
Acetylglucosamine
Cell Fusion
Glucosamine
Mannose
Fusion reactions
Clone Cells
CD55 Antigens
Methylnitronitrosoguanidine
Defects
K562 Cells
Sorting
Membrane Proteins
Genes
Cells
Availability

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mohney, R. P., Knez, J. J., Ravi, L., Sevlever, D., Rosenberry, T. L., Hirose, S., & Medof, M. E. (1994). Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants. Journal of Biological Chemistry, 269(9), 6536-6542.

Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants. / Mohney, Robert P.; Knez, Jansen J.; Ravi, Lakshmeswari; Sevlever, Daniel; Rosenberry, Terrone L.; Hirose, Shinichi; Medof, M. Edward.

In: Journal of Biological Chemistry, Vol. 269, No. 9, 04.03.1994, p. 6536-6542.

Research output: Contribution to journalArticle

Mohney, RP, Knez, JJ, Ravi, L, Sevlever, D, Rosenberry, TL, Hirose, S & Medof, ME 1994, 'Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants', Journal of Biological Chemistry, vol. 269, no. 9, pp. 6536-6542.
Mohney, Robert P. ; Knez, Jansen J. ; Ravi, Lakshmeswari ; Sevlever, Daniel ; Rosenberry, Terrone L. ; Hirose, Shinichi ; Medof, M. Edward. / Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 9. pp. 6536-6542.
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abstract = "Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992). J. Biol. Chem. 267, 5272- 5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway- defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine- inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan (EthN-P→)ManGlcN- and EthN-P-6Man(EthN-P- 6)Man(EthN-P→)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D- glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.",
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AU - Mohney, Robert P.

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AU - Ravi, Lakshmeswari

AU - Sevlever, Daniel

AU - Rosenberry, Terrone L.

AU - Hirose, Shinichi

AU - Medof, M. Edward

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N2 - Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992). J. Biol. Chem. 267, 5272- 5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway- defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine- inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan (EthN-P→)ManGlcN- and EthN-P-6Man(EthN-P- 6)Man(EthN-P→)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D- glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.

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