Structural and Functional Analysis of the Pig-a Protein That is Mutated in Paroxysmal Nocturnal Hemoglobinuria

Fapm, Edward R Norris, Md, Fapa,; Howard, Thad A.; Marcus, Stacy J.; Ware, Russell E.

doi:10.1006/bcmd.1997.0152

Cited by 7 publications

(6 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Other amino‐acid residues appear to be important for Gpi3p function. Mutagenesis studies of the human sequence homologue of Gpi3p, Pig‐A, have established that Gly48, His128, Ser129, and Ser155 are important for function [8,21], but mutations in the EX 7 E Glu residues were not examined in these studies.…”

Section: Discussionmentioning

confidence: 99%

Comparative importance in vivo of conserved glutamate residues in the EX₇E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Kostova

Yan

Vainauskas

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX 7 E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX 7 E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX 7 E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by sitespecific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25°C, but, whereas the E289D strain grew at 37°C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX 7 E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3 + and supported growth at 37°C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX 7 E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX 7 E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.

show abstract

Section: Discussionmentioning

confidence: 99%

Comparative importance in vivo of conserved glutamate residues in the EX₇E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Kostova

Yan

Vainauskas

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…b Predicted ef®ciency calculated by adding the values at the o, o 1 and o 2 sites and normalizing them as in a . acquired hemolytic disorder PNH, mutation of the affected gene termed PIG-A eventuates in a partial rather than a complete defect in GPIanchoring [Norris et al, 1997]. Mutated cells of these patients exhibit some but not other GPIanchored surface proteins.…”

Section: Discussionmentioning

confidence: 99%

Comparative efficiencies of C‐terminal signals of native glycophosphatidylinositol (GPI)‐anchored proproteins in conferring GPI‐anchoring

Chen

Knez

Merrick

et al. 2001

J of Cellular Biochemistry

View full text Add to dashboard Cite

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post-translational modification has a C-terminal GPI-anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed omega-->omega + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C-terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3' end-sequence encoding omega-2 to the C-terminus with the corresponding C-terminal sequences of nine other human GPI-anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS-PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out-performing the others in GPI-anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at omega-->omega + 2.

show abstract

“…The reader is referred to the associated web page (http:// mendel.imp.univie.ac.at/SEQUENCES/gpi-biosynthesis/) for discussion of the catalytic and ligand binding sites, (17,19) mutations, (20)(21)(22) isoforms, (23) and pseudogenes of PIG-A. (24) PIG-H/GPI15…”

Section: Pig-a/gpi3mentioning

confidence: 99%

Enzymes and auxiliary factors for GPI lipid anchor biosynthesis and post‐translational transfer to proteins

et al. 2003

View full text Add to dashboard Cite

GPI lipid anchoring is an important post-translational modification of eukaryote proteins in the endoplasmic reticulum. In total, 19 genes have been directly implicated in the anchor synthesis and the substrate protein modification pathway. Here, the molecular functions of the respective proteins and their evolution are analyzed in the context of reported literature data and sequence analysis studies for the complete pathway (http://mendel.imp.univie.ac.at/SEQUENCES/gpi-biosynthesis/) and questions for future experimental investigation are discussed. Studies of two of these proteins have provided new mechanistic insights. The cytosolic part of PIG-A/GPI3 has a two-domain alpha/beta/alpha-layered structure; it is suggested that its C-terminal subsegment binds UDP-GlcNAc whereas the N-terminal domain interacts with the phosphatidylinositol moiety. The lumenal part of PIG-T/GPI16 apparently consists of a beta-propeller with a central hole that regulates the access of substrate protein C termini to the active site of the cysteine protease PIG-K/GPI8 (gating mechanism) as well as of a polypeptide hook that embraces PIG-K/GPI8. This structural proposal would explain the paradoxical properties of the GPI lipid anchor signal motif and of PIG-K/GPI8 orthologs without membrane insertion regions in some species.

show abstract

Structural and Functional Analysis of the Pig-a Protein That is Mutated in Paroxysmal Nocturnal Hemoglobinuria

Cited by 7 publications

References 32 publications

Comparative importance in vivo of conserved glutamate residues in the EX₇E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Comparative importance in vivo of conserved glutamate residues in the EX₇E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Comparative efficiencies of C‐terminal signals of native glycophosphatidylinositol (GPI)‐anchored proproteins in conferring GPI‐anchoring

Enzymes and auxiliary factors for GPI lipid anchor biosynthesis and post‐translational transfer to proteins

Contact Info

Product

Resources

About

Structural and Functional Analysis of the Pig-a Protein That is Mutated in Paroxysmal Nocturnal Hemoglobinuria

Cited by 7 publications

References 32 publications

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Comparative efficiencies of C‐terminal signals of native glycophosphatidylinositol (GPI)‐anchored proproteins in conferring GPI‐anchoring

Enzymes and auxiliary factors for GPI lipid anchor biosynthesis and post‐translational transfer to proteins

Contact Info

Product

Resources

About

Comparative importance in vivo of conserved glutamate residues in the EX₇E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly

Comparative importance in vivo of conserved glutamate residues in the EX₇E motif retaining glycosyltransferase Gpi3p, the UDP‐GlcNAc‐binding subunit of the first enzyme in glycosylphosphatidylinositol assembly