O-GlcNAcylation determines the function of the key O-GalNAc glycosyltransferase C1GalT1 in bladder cancer

Abstract

Protein glycosylation is a type of protein post-translational modification. One specific example is the modification of proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc). Enhanced levels of both O-GalNAc and O-GlcNAc in bladder cancer (BlCa) have been reported previously. However, the interplay between O-GalNAc and O-GlcNAc has yet to be explored. Herein, we find that the expression level of core1 β-1,3-galactosyltransferase (C1GalT1), which is responsible for extending and maturing mucin-type O-glycans, is increased in BlCa. This increase is accompanied by O-GlcNAc modification of C1GalT1. This modification stabilizes C1GalT1 expression and strengthens its interaction with its chaperone Cosmc. Mutation at Thr229 or Thr233 attenuates C1GalT1 stability and facilitates its degradation via the proteasome pathway. Furthermore, a decrease in C1GalT1 inhibits the pro-tumorigenic effect on bladder cancer cells by suppressing glycolysis.

1. Introduction

Protein glycosylation is a widespread post-translational modification that affects more than 70% of all human proteins [1]. This modification plays a key role in molecular recognition and cell‒cell adhesion, and its dysregulation can result in a variety of developmental defects, growth disorders, and lethal diseases. In the context of malignant transformation, abnormal protein glycosylation frequently occurs, leading to the expression of specific tumor-associated glycans [2]. Atypical glycans on cancer cells are commonly linked to tumor grade, invasiveness, and metastatic potential, and they are correlated with unfavorable overall prognoses [3,4]. The linkage of glycan chains to polypeptide backbones in glycoproteins commonly occurs via the nitrogen of Asn residues (termed N-glycans) or the oxygen of Ser/Thr residues (termed O-glycans), with O-glycans further subdivided into two types: O-GlcNAcylation (O-linked β-N-acetylglucosamine, O-GlcNAc) and O-GalNAcylation (O-linked α-N-acetylgalactosamine, O-GalNAc) [5].

Polypeptide GalNActransferases (ppGalNAcTs) catalyze the addition of GalNAc to Ser/Thr to generate the Tn antigen (GalNAcα-O-Ser/Thr), which represents the initial form of O-GalNAc glycans [6]. Subsequent sequential glycosyltransferases extend and branch O-GalNAc glycans based on the Tn antigen. Core1 β-1,3-galactosyltransferase (C1GalT1, also known as T-synthase) transfers Gal to GalNAc to form Gal-β-1-3GalNAcα1-Ser/Thr (referred to as the T antigen), which serves as a precursor for the subsequent elongation and maturation of mucin-type O-glycans [7]. Truncated T and Tn antigens are prominently expressed in various types of cancers [8]. O-GlcNAc is modified by O-GlcNAc transferase (OGT), which adds O-GlcNAc, and O-GlcNAcase (OGA), which removes O-GlcNAc. However, the GlcNAc structure is typically not further modified or elongated to generate more complex structures [9]. O-GalNAc and O-GlcNAc glycosylation play crucial roles in various cellular processes, such as protein folding, localization, and degradation, as well as cellular signaling and immune responses. Moreover, anomalous O-GalNAc and O-GlcNAc glycosylation has been implicated in diverse diseases, including cancers, neurodegenerative illnesses, and autoimmune disorders [10,11].

Bladder cancer (BlCa) is a frequently occurring malignancy in humans, with a fifth-place rank in terms of incidence and a continuously increasing frequency over the past decade. The non-muscle-invasive form of BlCa is prevalent in more than 70% of patients, while approximately 25% of patients are initially identified as having the muscle-invasive type. Muscle-invasive bladder cancer patients have a 50% chance of developing distant metastases and a grim prognosis [12]. Abnormal O-GalNAc glycosylation, including modifications in the T antigen and Tn antigen expression, has been reported in BlCa [13,14]. Additionally, an amplified level of O-GlcNAc has also been detected in BlCa [15]. However, investigations on the interaction between O-GalNAc and O-GlcNAc in the context of BlCa have not been conducted thus far.