Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain

Abstract

Many eukaryotic proteins are anchored to the cell surface via the glycolipid glycosylphosphatidylinositol (GPI). Mammalian GPIs have a conserved core but exhibit diverse N-acetylgalactosamine (GalNAc) modifications, which are added via a yet unresolved process. Here we identify the Golgi-resident GPI-GalNAc transferase PGAP4 and show by mass spectrometry that PGAP4 knockout cells lose GPI-GalNAc structures. Furthermore, we demonstrate that PGAP4, in contrast to known Golgi glycosyltransferases, is not a single-pass membrane protein but contains three transmembrane domains, including a tandem transmembrane domain insertion into its glycosyltransferase-A fold as indicated by comparative modeling. Mutational analysis reveals a catalytic site, a DXD-like motif for UDP-GalNAc donor binding, and several residues potentially involved in acceptor binding. We suggest that a juxtamembrane region of PGAP4 accommodates various GPI-anchored proteins, presenting their acceptor residue toward the catalytic center. In summary, we present insights into the structure of PGAP4 and elucidate the initial step of GPI-GalNAc biosynthesis.

Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

“…1a, bottom), i.e., T5 mAb does not bind free GPI when GalNAc is galactosylated (Fig. 1a, middle) 11, 32, 33 . Therefore, T5 mAb is useful to identify mutant cells defective in galactosylation of GPI-GalNAc.…”

“…4c), indicating that enzymatic activity of UGCG is required for galactosylation of GPI-GalNAc. To determine whether the requirement for UGCG for galactosylation of GPI-GalNAc is a general phenomenon, we used CHO cells because we previously reported that CHO cells express free GPI bearing a galactosylated GalNAc side-chain as well as GPI-APs 11 . Knockout of UGCG from CHO cells also made T5-staining positive without affecting the GS-II lectin staining profile (Supplementary Fig.…”

“…To analyze the GPI structures derived from HFGF-CD59, the bands of purified HFGF-CD59 were excised, reduced with 10 mM dithiothreitol (DTT) and alkylated with 55 mM iodoacetamide. After in-gel digestion with trypsin, the resultant peptides were subjected to mass spectrometry analyses on LC-ESI system as previously described 11 . Briefly, LC-MS/MS analysis was performed in the positive ion mode on a nanoLC system (Advance, Michrom BioRescources) using a C18 column (0.1 × 150 mm) coupled to an LTQ Qrbitrap Velos mass spectrometer (Thermo Scientific).…”

“…Structural studies of GPIs from some mammalian GPI-APs indicated that the first mannose (Man1) is often modified with β1,4-linked N -acetylgalactosamine (GalNAc) 9, 10 . This modification is mediated by PGAP4 (Post-GPI attachment to proteins 4 also known as TMEM246), a recently identified Golgi-resident, GPI-specific GalNAc-transferase 11 . The GalNAc side-chain can be further modified with β1,3 galactose (Gal) by an unknown galactosyltransferase (Gal-T) and then with sialic acid (Sia) (Fig.…”

Mutations in PIGU Impair the Function of the GPI Transamidase Complex, Causing Severe Intellectual Disability, Epilepsy, and Brain Anomalies

“…Use of the T5 4E10 antibody allowed detection of free GPI anchors. [15][16][17][18] Relative reduction of GPI-AP was calculated as a ratio of the staining index (SI) of an affected individual to the SIs of healthy parents and healthy unrelated controls. It is noteworthy that there were only subtle differences in GPI-AP staining between heterozygous carriers of pathogenic mutations (parents) and unrelated healthy controls.…”