Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

Abstract

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.

Phthalocyanines and Tetrapyrazinoporphyrazines with Two Cationic Donuts: High Photodynamic Activity as a Result of Rigid Spatial Arrangement of Peripheral Substituents

“…The localization was further confirmed for the most active Pc 10 by staining with another specific probe (ER-Tracker) together with various organelle-specific protein transfections (Lamp1-RFP for lysosomes, Rab7a-GFP for late endosomes, and GALNT2-RFP for the Golgi apparatus) (Figure ). The punctuate fluorescence of 10 was exclusively colocalized with markers for the endolysosomal compartment (Lamp1 and Rab7a; Figure E,F).…”

An Integrated Mass Spectroscopy Data Processing Strategy for Fast Identification, In-Depth, and Reproducible Quantification of Protein O-Glycosylation in a Large Cohort of Human Urine Samples

“…First, intact glycopeptide identification, which provides both the peptide sequence and the corresponding glycan composition simultaneously, is increasingly accepted in glycoproteome studies. However, unlike N -glycosylation, which occurs in NXS/T/C amino acid motifs, there are no sequence motifs on the substrate proteins for O -glycan attachment. − Therefore, each serine and threonine residue must be considered as a potential modification site in O -glycoproteome database searching. The 1.5 million serine and threonine residues in the mammalian proteome and the tens of glycoforms of mammalian O -glycans as variable modifications result in countless combinations and extremely large searching space for intact O -glycopeptide database searching.…”

CREB3L2 Modulates Nerve Growth Factor-Induced Cell Differentiation

“…In agreement with the increase of fluorescence intensity, Golgi volume increases ∼1.5-to 2-fold after 4 and 6 days of NGF treatment (Figure 1B). Moreover, we analyzed the fluorescence intensity of other proteins associated with membrane trafficking, such as the GTPase Rab1b, essential for ER to Golgi transport, localized at the ER-Golgi-Intermediate compartment (Garcia et al, 2011;Slavin et al, 2011) and the medial-and trans-Golgi marker GalNAc-T2 (Rottger et al, 1998). As shown in Figures 1C-E, the fluorescence intensity of both protein markers correlates with the increase of the GM130 signal.…”