Brain Contains HNK-1 Immunoreactive O-Glycans of the Sulfoglucuronyl Lactosamine Series that Terminate in 2-Linked or 2,6-Linked Hexose (Mannose)

Chun-Ting Yuen,Wengang Chai,R Wendy Loveless,Alexander M Lawson,Richard U Margolis,Ten Feizi

doi:10.1074/jbc.272.14.8924

Chun-Ting Yuen, Wengang Chai + Show 4 more

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https://doi.org/10.1074/jbc.272.14.8924

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Abstract

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells also binds to selected regions in nervous tissue. The antigen is a carbohydrate that has attracted much interest as its expression is developmentally regulated in nervous tissue, and it is found, and proposed to be a ligand, on several of the adhesive glycoproteins of the nervous system. It is also expressed on glycolipids and proteoglycans, and is the target of monoclonal auto-antibodies that give rise to a demyelinating disease. The epitope, as characterized on glycolipids isolated from the nervous system, is expressed on 3-sulfated glucuronic acid joined by beta1-3-linkage to a neolacto backbone. Here we exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides. The immunoreactive oligosaccharides detected are tetra- to octasaccharides that are very minor components among a heterogeneous population, each representing less than 0.1% of the starting material. Their peripheral and backbone sequences resemble those of the HNK-1-positive glycolipids. An unexpected finding is that they terminate not with N-acetylgalactosaminitol but with hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as 2-substituted mannitol.

Highlights

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells binds to selected regions in nervous tissue
We exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides
Alditols (Table III), indicate that these HNK-1 immunoreactive hexa- and octasaccharides are a population of polylactosaminetype glycans terminating in 2-substituted hexitol which upon oxidation is cleaved to produce remnants of the reduced hexitol with three, four, or five carbons (Scheme 1, Table IV). This investigation illustrates the power of the neoglycolipid technology to detect and biochemically characterize bioactive oligosaccharides that are extremely minor components of a heterogeneous population

Summary

Introduction

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells binds to selected regions in nervous tissue. HNK-1 antigen, as characterized biochemically on glycolipids, is expressed on 3-sulfated glucuronic acid joined by ␤1-3-linkage to a (poly)Nacetyllactosamine backbone (8 –10) This carbohydrate sequence has been implicated as a ligand for the leukocyteendothelium adhesion molecules, L- and P-selectins [16, 17] and for a number of cell-interaction systems in nervous tissue [11, 18], notably as a ligand for the major glycoprotein of myelin, Po, in the peripheral nervous system [19], the extracellular matrix protein, laminin [20], and an adhesive protein, amphoterin, of the cerebellum [21]. Having observed immunoreactive oligosaccharides among all three oligosaccharide populations, a more detailed investigation has been performed of oligosaccharides released by reductive alkaline hydrolysis

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