Synthetic HNK-1 containing glycans provide insight into binding properties of serum antibodies from MAG-neuropathy patients

Abstract

Anti-myelin-associated glycoprotein (anti-MAG) neuropathy is an autoimmune disease in which IgM autoantibodies target glycoconjugates of peripheral nerves resulting in progressive demyelination. To examine fine specificities of serum IgM autoantibodies and develop a more robust platform for diagnosis and disease monitoring, we describe here a chemoenzymatic approach that readily provided a panel of HNK-1 containing oligosaccharides presented on type 2 oligo-N-acetyl lactosamine (LacNAc) chains typical of glycosphingolipids. The compounds were prepared by a chemoenzymatic strategy in which an oligo-LacNAc structure was assembled enzymatically and then subjected to protecting group manipulation to chemically install a 3-O-sulfate glucuronic acid moiety. The synthetic strategy is highly divergent and made it possible to prepare from key precursors, additional compounds lacking sulfate of HNK-1 and derivatives in which the HNK-1 epitope is replaced by sulfate or sialic acid. The oligosaccharides were printed as a microarray to examine binding specificities of several monoclonal antibodies and serum antibodies of anti-MAG neuropathy patients. Surprisingly, three distinct patient subgroups were identified with variable dependance on the length of the LacNAc chain and sulfation of the glucuronyl moiety. In most cases, a lacto-neohexaose backbone was required for binding indicating the antibodies target corresponding glycosphingolipids. Significance statementA chemoenzymatic strategy is introduced in which a glycan backbone is assembled by glycosyltransferases to give a core oligosaccharide that is subjected to protecting group manipulations and chemical glycosylations to install terminal epitopes. It addresses limitations of enzymatic synthesis when specific glycosyltransferases or glycan-modifying enzymes for terminal epitope synthesis are not readily available. It provided an unprecedented panel of HNK-1 containing oligosaccharides, which was used to develop a glycan microarray that uncovered distinct binding preferences of serum antibodies of anti-MAG patients. The clinical spectrum of IgM monoclonal gammopathy varies substantially and an understanding of binding properties of IgM auto-antibodies will provide opportunities to monitor disease progression and develop personalized treatment options.