Development of a Novel Method for Analyzing Collagen O-glycosylations by Hydrazide Chemistry

Abstract

In recent years, glycopeptide purification by hydrazide chemistry has become popular in structural studies of glycoconjugates; however, applications of this method have been almost completely restricted to analysis of the N-glycoproteome. Here we report a novel method for analyzing O-glycosylations unique to collagen, which are attached to hydroxylysine and include galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine. We established a hydrazide chemistry-based glycopeptide purification method using (1) galactose oxidase to introduce an aldehyde into glycopeptides and (2) formic acid with heating to elute the bound glycopeptides by cleaving the hydrazone bond. This method allows not only identification of O-glycosylation sites in collagen but also concurrent discrimination of two types of carbohydrate substitutions. In bovine type I and type II collagens, galactosyl-hydroxylysine /glucosyl-galactosyl-hydroxylysine -containing peptides were specifically detected on subsequent comprehensive liquid chromatography (LC)/MS analysis, and many O-glycosylation sites, including unreported ones, were identified. The position of glycosylated hydroxylysine, which is determined by our unambiguous and simple method, could provide insight into the physiological role of the modifications. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010397, 1-9, 2012. Galactosyl-hydroxylysine (GHL)1 and glucosyl-galactosylhydroxylysine (GGHL) are O-glycosylations unique to collagen (1). They are also found in several other proteins having a collagen-like sequence, such as the C1q complement protein (2). A monosaccharide or disaccharide is attached to the hydroxylysine residue lying in the Y position of repeating collagenous Gly-X-Y triplets within a triple helix. Specific enzymes add the carbohydrates to hydroxylysine before triple helix formation in the endoplasmic reticulum (3, 4). The carbohydrate content varies with collagen type, tissue, and physiological conditions. There are few glycosylated hydroxylysines in fibrillar-forming collagens. For example, type I collagen alpha 1 chain has approximately one residue per 1000 amino acid residues, the alpha 2 chain has approximately two residues, and type II collagen alpha 1 chain has ϳ10 residues (5). In contrast, most lysine residues are glycosylated in network-forming collagens such as type IV collagen. Although disorder-related alterations, such as overglycosylation in osteogenesis imperfecta (6) and spondyloepiphyseal dysplasia (7), have been reported, the biological significance of the carbohydrates remains unclear. The position of glycosylated hydroxylysine in a primary amino acid sequence could provide insight into the physiological role of the modifications.In the 1970s, extensive structural studies revealed nearly all the primary amino acid sequences of major collagens by automated Edman degradation on a protein sequencer, but a few sites have remained uncertain because of some modifications (8). When the collagen peptides, which are digested by cyanogen bromide (CNBr) ...

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

“…Both of the aforementioned O-linked glycosylations are unique to fibrillar collagens (63) and have been previously reported in other proteomics datasets (60,62,71,72). Although their biological function still remains somewhat unclear (73), they are believed to play a role in modulating the structural stability of collagen (74,75).…”

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

“…Analysis of Glycosylation-To verify the presence of sugar modifications, the glycosylated peptides were isolated using hydrazide chemistry (27). Galactose oxidase was bound to Sepharose 4B under basic conditions.…”

TFE3, HSP47, and CREB3 Pathways of the Mammalian Golgi Stress Response

“…Because collagens receive mucin type Oglycosylation (Taga et al, 2012), it is possible that a small amount of HSP47 transiently translocates to the Golgi, where HSP47 assists mucin type O-glycosylation of collagens. The link between HSP47 and the Golgi as well as the molecular mechanism of the HSP47 pathway are important issues for future analyses.…”