Anti-myelin associated glycoprotein antibodies recognize HNK-1 epitope on CNS

Abstract

Antibodies to myelin-associated glycoprotein (MAG) are associated with demyelinating polyneuropathy and are specific for the HNK-1 epitope. To test if anti-MAG IgM recognize HNK-1 on CNS, sera from 20 patients and 238 controls were tested on rat slices by indirect immunofluorescence (IIF). IgM from anti-MAG positive patients, but not from control sera, stained rat brain with perineuronal or neuropil pattern, depending on the CNS region. IIF titers significantly correlated with ELISA anti-MAG titers. The staining of patients’ sera were inhibited by mouse anti-HNK-1 monoclonal antibody. Our results demonstrate that anti-MAG IgM recognizes HNK-1 outside the peripheral nerve myelin carriers.

Introduction

Serum antibody specific for myelin-associated glycoprotein (MAG) is often found in patients with IgM monoclonal gammopathy (MG) and slowly progressive, demyelinating, sensory-motor polyneuropathy (Braun et al., 1982, Quarles and Weiss, 1999). MAG, a large molecule of the nervous system, is concentrated in periaxonal Schwann-cell membranes and paranodal loops of the peripheral nerve myelin, where it acts as an adhesion molecule for interactions between Schwann cells and axons (Bollensen and Schachner, 1987, Hammer et al., 1993). MAG structure consists of five immunoglobulin-like domains and a carbohydrate epitope, HNK-1, which has been demonstrated to be the specific epitope targeted by anti-MAG IgM in patients with polyneuropathy (Ilyas et al., 1984, Burger et al., 1992). As a matter of fact, anti-MAG positive sera do not react with deglycosylated MAG (Nobile-Orazio et al., 1984), while they stain neuroblastoma cell lines that express the HNK-1 epitope but not the MAG protein (Isoardo et al., 2005). In clinical practice, anti-MAG antibody detection assays (ELISA and Western Blot, WB) usually utilize MAG purified from bovine brain or human autopsy material (Nobile-Orazio et al., 1989, Kuijf et al., 2009). These methods are quite expensive and time-consuming, and often lead to conflicting results (Kuijf et al., 2009). MAG from rodent myelin is not suitable for anti-MAG testing (O'Shannessy et al., 1985), probably because of a different antigenic conformation or, more likely, because rodent MAG lacks the HNK-1 epitope (Chou and Jungalwala, 2001). However, there is evidence that HNK-1 is abundantly expressed in the CNS gray matter of many species, including humans (see Morita et al., 2008 for review).

HNK-1 consists of a 3’-sulfated glucuronic acid attached to C3 of a N-acetyllactosamine unit (Chou et al., 1986). First identified as a marker of the natural killer cells by a hybridoma antibody, HNK-1 was later found to be expressed by adhesion molecules of the immunoglobulin superfamily (such as MAG, P0, NCAM, transiently expressed axonal glycoprotein-1, F3/F11/contactin), by members of the tenascin family, by integrins, proteoglycans, and by acidic glycolipids of the nervous system, such as sulfate-3-glucuronyl paragloboside (SGPG) (Bollensen and Schachner, 1987, Hammer et al., 1993, Chou and Jungalwala, 2001, Morita et al., 2008). In the brain many of these molecules are components of the grey matter extracellular matrix; they can be either dispersed diffusely throughout the neuropil, or concentrated to form lattice-like aggregates, the so-called perineuronal nets. Nets are located around cell bodies, dendrites and axon initial segments of many neurons, especially those expressing the calcium-binding protein parvalbumin (Kosaka et al., 1990, Celio and Blümcke, 1994).

We investigated the reactivity of IgM from polyneuropathy patients by using indirect immunofluorescence (IIF) on rat CNS. Aim of our study was to test whether this antibody can recognize HNK-1 epitope outside its peripheral nerve myelin carriers, and to evaluate if IIF on rat CNS may be used as a screening method for the identification of anti-MAG antibodies.