N-Glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures

Abstract

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.

Sialylation of 1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

“…Several other cell-associated ligands for gal-3 have been reported, including the epidermal growth factor receptor (52), transforming growth factor-␤ receptor (52,53), T cell receptor (53), Mac-2-binding protein (54), RAGE (32), Lamp-1,2, and carcinoembryonic antigen (55). Interestingly, carcinoembryonic antigen is highly expressed in colon cancer cells, and like the ␤1 integrin, is known to be hypersialylated with ␣2-6 sialic acid (56). Additional experiments will be needed to identify other galectin-binding proteins and also to dissect the specific galectin-dependent functions of these respective molecules.…”

Inhibition of Mammalian Glycan Biosynthesis Produces Non-self Antigens for a Broadly Neutralising, HIV-1 Specific Antibody

“…[27][28][29][30] The effect of kifunensine treatment on the 15 N-linked carbohydrate structures of CEACAM1 was determined by release of the total N-linked glycan pool using PNGase F and subsequent time of flight (TOF) and fragmentation (MS/MS) analysis by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS) of both CEACAM1 and CEACAM1 kif (Figure 2). The range of complex-type glycans observed on the untreated sample is consistent with previous reports for this glycoprotein 29 (Figure 2(a)). However, expression in the presence of kifunensine (20 μM) led to a dramatic reduction in glycan complexity towards a single, abundant oligomannose-type glycoform (Figure 2(b)).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 1999–2000

“…There have been several reports of the identification of sialylated glycans containing more sialic acid residues than would be predicted from the number of antennae. Glycoproteins affected are rat liver cell-cell adhesion molecule (C-CAM) (Kannicht et al, 1999), human CD52 (Schröter et al, 1999), and the major urinary protein from the house mouse (Mus musculus) where the extra sialic acid a-(2 ! 3)-linked to GlcNAc residues .…”