LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

Abstract

Both LARGE1 (formerly LARGE) and its paralog LARGE2 are bifunctional glycosyltransferases with xylosy- and glucuronyltransferase activities, and are capable of synthesizing polymers composed of a repeating disaccharide [-3Xylα1,3GlcAβ1-]. Post-translational modification of the O-mannosyl glycan of α-dystroglycan (α-DG) with the polysaccharide is essential for it to act as a receptor for ligands in the extracellular matrix (ECM), and both LARGE paralogs contribute to the modification in vivo. LARGE1 and LARGE2 have different tissue distribution profiles and enzymatic properties; however, the functional difference of the homologs remains to be determined, and α-DG is the only known substrate for the modification by LARGE1 or LARGE2. Here we show that LARGE2 can modify proteoglycans (PGs) with the laminin-binding glycan. We found that overexpression of LARGE2, but not LARGE1, mediates the functional modification on the surface of DG−/−, Pomt1−/− and Fktn−/− embryonic stem cells. We identified a heparan sulfate-PG glypican-4 as a substrate for the LARGE2-dependent modification by affinity purification and subsequent mass spectrometric analysis. Furthermore, we showed that LARGE2 could modify several additional PGs with the laminin-binding glycan, most likely within the glycosaminoglycan (GAG)-protein linkage region. Our results indicate that LARGE2 can modify PGs with the GAG-like polysaccharide composed of xylose and glucuronic acid to confer laminin binding. Thus, LARGE2 may play a differential role in stabilizing the basement membrane and modifying its functions by augmenting the interactions between laminin globular domain-containing ECM proteins and PGs.

Cancer Cell Acid Adaptation Gene Expression Response Is Correlated to Tumor-Specific Tissue Expression Profiles and Patient Survival

“…Other highly upregulated genes included interferon-induced transmembrane protein 1 ( IFITM1 ), which, together with other interferon-regulated genes, are highly upregulated in several cancers and assigned key roles in driving aggressiveness and chemotherapy resistance [ 34 , 35 ]. In addition, the genes encoding Sushi-containing Domain-3 (SUSD3), an estrogen-regulated membrane-localized protein previously found to be upregulated by chronic acidosis [ 15 ] and to play a key role in breast cancer cell migration [ 36 ], and LARGE2 , a bifunctional glycosyltransferase involved in proteoglycan modification and hence in cell–extracellular matrix (ECM) interaction [ 37 ], were among the 10 most upregulated genes ( Figure 2 A).…”

Wnt-driven LARGE2 mediates laminin-adhesive O-glycosylation in human colonic epithelial cells and colorectal cancer

“…In this model, addition of DOX for 72 h led to~10-25 fold higher levels of LARGE2 mRNA when compared to Wnt-active SW480 and PDTO1 cells or Wnt-activated HT-29 cells (Additional file 6C). Since ectopic LARGE2 has been reported to also O-glycosylate laminin-binding and IIh6c4-reactive glypican 4 (GPC4) in mouse ES cells [55], we performed quantitative mass spectrometry (LC-MS/MS) analysis on WGA-AE purified glycoproteins from these cells. Due to the shift in molecular weight (MW) of proteins upon O-glycosylation by LARGE2, we fractionated electrophoretically separated samples into 6 MW windows prior to LC-MS/MS analysis (Additional file 6D).…”

Laminin G-like domains: dystroglycan-specific lectins

“…For these reasons, it would make sense to consider matriglycan as a type of GAG, even though its disaccharide repeat does not contain an amino sugar. Interestingly, there may be overlap between the two types of modification: LARGE2 (the paralogue of LARGE) is able to attach matriglycan chains to proteoglycans, most likely by extending the GAG tetrasaccharide linkers [31•].…”