scholarly journals Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5462
Author(s):  
Si-Si Liu ◽  
Fei Jin ◽  
Yi-Shi Liu ◽  
Yoshiko Murakami ◽  
Yukihiko Sugita ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Posttranslational Modification ◽  
Homologous Proteins ◽  
Gpi Anchor ◽  
Purification Method ◽  
C Terminus ◽  
Precursor Proteins ◽  
Amino Acid Mutations ◽  
Gpi Transamidase ◽  
Particle Images

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

scholarly journals Gaa1p and Gpi8p Are Components of a Glycosylphosphatidylinositol (GPI) Transamidase That Mediates Attachment of GPI to Proteins

2000 ◽  
Vol 11 (5) ◽  
pp. 1523-1533 ◽  
Author(s):  
Kazuhito Ohishi ◽  
Norimitsu Inoue ◽  
Yusuke Maeda ◽  
Junji Takeda ◽  
Howard Riezman ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Posttranslational Modification ◽  
Eukaryotic Cell ◽  
Surface Proteins ◽  
F9 Cells ◽  
Precursor Proteins ◽  
Key Enzyme ◽  
Gpi Transamidase

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted aGAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.

scholarly journals Function of AtPGAP1 in GPI anchor lipid remodeling and transport to the cell surface of GPI-anchored proteins

2021 ◽  
Author(s):  
César Bernat-Silvestre ◽  
Judit Sanchez-Simarro ◽  
Yingxuan Ma ◽  
Kim Johnson ◽  
Fernando Aniento ◽  
...  
Keyword(s):  
Cell Surface ◽  
Acyl Chain ◽  
Growth Stress ◽  
Loss Of Function ◽  
Gpi Anchor ◽  
C Terminus ◽  
Gpi Transamidase ◽  
Er Export ◽  
Inositol Ring

ABSTRACTGPI-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signalling and cell wall biosynthesis. The GPI-anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodelled. In mammals and yeast, this remodelling is required for GPI-APs to be included in Coat Protein II (COPII) coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodelling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and PGAP1 (mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis PGAP1 localizes to the ER and probably functions as the GPI inositol-deacylase which cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs.One sentence summaryGPI anchor lipid remodeling in GPI-anchored proteins is required for their transport to the cell surface in Arabidopsis.

scholarly journals TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1256-1262 ◽  
Author(s):  
Thomas J. Girard ◽  
Elodee Tuley ◽  
George J. Broze
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Feedback Inhibition ◽  
Culture Media ◽  
Factor Viia ◽  
Factor Xa ◽  
Gpi Anchor ◽  
C Terminus ◽  
Before And After ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

Functional analysis and molecular model of the human urate transporter/channel, hUAT

2002 ◽  
Vol 283 (1) ◽  
pp. F150-F163 ◽  
Author(s):  
Edgar Leal-Pinto ◽  
B. Eleazar Cohen ◽  
Michael S. Lipkowitz ◽  
Ruth G. Abramson
Keyword(s):  
Functional Analysis ◽  
Lipid Bilayers ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Molecular Model ◽  
Open Probability ◽  
Homologous Proteins ◽  
Urate Transporter ◽  
Binding Domains

Recombinant protein, designated hUAT, the human homologue of the rat urate transporter/channel (UAT), functions as a highly selective urate channel in lipid bilayers. Functional analysis indicates that hUAT activity, like UAT, is selectively blocked by oxonate from its cytosolic side, whereas pyrazinoate and adenosine selectively block from the channel's extracellular face. Importantly, hUAT is a galectin, a protein with two β-galactoside binding domains that bind lactose. Lactose significantly increased hUAT open probability but only when added to the channel's extracellular side. This effect on open probability was mimicked by glucose, but not ribose, suggesting a role for extracellular glucose in regulating hUAT channel activity. These functional observations support a four-transmembrane-domain structural model of hUAT, as previously predicted from the primary structure of UAT. hUAT and UAT, however, are not functionally identical: hUAT has a significantly lower single-channel conductance and open probability is voltage independent. These differences suggest that evolutionary changes in specific amino acids in these highly homologous proteins are functionally relevant in defining these biophysical properties.

GAA1 recognizes the GPI anchor attachment signals and is complexed with GP18 to form a GPI transamidase

1998 ◽  
Vol 35 (6-7) ◽  
pp. 388 ◽  
Author(s):  
K. Ohishi ◽  
N. Inoue ◽  
J. Takeda ◽  
D. Hamburger ◽  
H. Riezman ◽  
...  
Keyword(s):  
Gpi Anchor ◽  
Gpi Transamidase

scholarly journals Single‐residue posttranslational modification sites at the N‐terminus, C‐terminus or in‐between: To be or not to be exposed for enzyme access

PROTEOMICS ◽  
2015 ◽  
Vol 15 (14) ◽  
pp. 2525-2546 ◽  
Author(s):  
Fernanda L. Sirota ◽  
Sebastian Maurer‐Stroh ◽  
Birgit Eisenhaber ◽  
Frank Eisenhaber
Keyword(s):  
Posttranslational Modification ◽  
C Terminus ◽  
N Terminus

scholarly journals Functional Domains of Murine Cytomegalovirus Nuclear Egress Protein M53/p38

2006 ◽  
Vol 80 (1) ◽  
pp. 73-84 ◽  
Author(s):  
Mark Lötzerich ◽  
Zsolt Ruzsics ◽  
Ulrich H. Koszinowski
Keyword(s):  
Binding Site ◽  
Random Mutagenesis ◽  
Murine Cytomegalovirus ◽  
Homologous Proteins ◽  
Herpes Simplex Virus 1 ◽  
Nuclear Egress ◽  
C Terminus ◽  
Localization Signal ◽  
Definition Of ◽  
Simplex Virus

ABSTRACT Two conserved herpes simplex virus 1 proteins, UL31 and UL34, form a complex at the inner nuclear membrane which governs primary envelopment and nuclear egress of the herpesvirus nucleocapsids. In mouse cytomegalovirus, a member of the betaherpesvirus subfamily, the homologous proteins M53/p38 and M50/p35 form the nuclear egress complex (NEC). Since the interaction of these proteins is essential for functionality, the definition of the mutual binding sites is a prerequisite for further analysis. Using a comprehensive random mutagenesis procedure, we have mapped the M53/p38 binding site of M50/p35 (A. Bubeck, M. Wagner, Z. Ruzsics, M. Lötzerich, M. Iglesias, I. R. Singh, and U. H. Koszinowski, J. Virol. 78:8026-8035). Here we describe a corresponding analysis for the UL31 homolog M53/p38. A total of 72 individual mutants were reinserted into the genome to test the complementation of the lethal M53 null phenotype. The mutants were also studied for colocalization and for coprecipitation with M50/p35. The analysis revealed that the nonconserved N-terminal one-third of M53/p38 provides the nuclear localization signal as an essential function. The collective results for many mutants localized the binding site for M50/p35 to amino acids (aa) 112 to 137. No single aa exchange for alanine could destroy NEC formation, but virus attenuation revealed a major role for aa K128, Y129, and L130. The lethal phenotype of several insertion and stop mutants indicated the functional importance of the C terminus of the protein.

scholarly journals Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

2006 ◽  
Vol 395 (2) ◽  
pp. 443-448 ◽  
Author(s):  
Patrick A. Lewis ◽  
Francesca Properzi ◽  
Kanella Prodromidou ◽  
Anthony R. Clarke ◽  
John Collinge ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Rocky Mountain ◽  
Brain Homogenate ◽  
Mouse Bioassay ◽  
Gpi Anchor ◽  
Glycosylphosphatidylinositol Anchor ◽  
Prion Infectivity ◽  
C Terminus ◽  
Prion Propagation

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrPSc, an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrPC), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrPSc to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrPC. Removal of the GPI anchor from PrPSc requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrPSc in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrPSc, (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrPSc or on prion infectivity.

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