The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase

The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase

^*Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106; and ^‡The Dana Institute, Drew University, Madison, NJ 07940

Contributed by Sidney Udenfriend

Abstract

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5′ extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans ≈25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Footnotes

↵ † Present address: Department of Pathology, Emory University, Atlanta, GA.
↵ § To whom reprint requests should be addressed at: Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF022913).
↵ ¶ This work was presented in abstract form at the 17th International Congress of Biochemistry and Molecular Biology (FASEB), Aug. 24–29, 1997, San Francisco.
ABBREVIATIONS:

GPI,

glycosylphosphatidylinositol;

ER,

endoplasmic reticulum;

HDZ,

hydrazine;

DAF,

decay-accelerating factor;

PI,

phosphatidylinositol;

PLAP,

placental alkaline phosphatase;

PI-PLC,

PI-specific phospholipase C;

TEA,

triethanolamine;

Neo,

neomycin