Development of smart anti-glycan reagents using immunized lampreys

Abstract

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. We identified a wide variety of glycan-specific VLRBs detectable in lamprey plasma after immunization with whole fixed cells, tissue homogenates, and human milk. The cDNAs from lamprey lymphocytes were cloned into yeast surface display (YSD) libraries for enrichment by multiple methods. We generated VLRB-Ig chimeras, termed smart anti-glycan reagents (SAGRs), whose specificities were defined by microarray analysis and immunohistochemistry. 15 VLRB antibodies were discovered that discriminated between linkages, functional groups and unique presentations of the terminal glycan motif. The development of SAGRs will enhance future studies on glycan expression by providing sequenced, defined antibodies for a variety of research applications.

Towards structure-focused glycoproteomics

“…For example, the recombinant mouse anti-Tn IgM ReBaGs6 was used to immunoprecipitate Tn-containing glycoproteins from Tn-positive Colo205 SimpleCells lysates for downstream mass spectrometry identification [ 154 ]. Smart anti-glycan reagents (SAGRs) represent another exciting type of emerging affinity tools [ 156 , 157 ]. SAGRs are chimeric proteins comprising murine IgG Fc and the single-chain variable lymphocyte receptors (VLRs) secreted by Petromyzon marinus VLRB + lymphocytes (B-like cell) upon immunisation with different glycoconjugates, cells and tissues of interest.…”

Integrated analysis of glycan and RNA in single cells

“…In terms of glycan-detection probes, cost-effective and commercially-available lectins, either from natural sources or recombinantly-expressed, were used for Glycan-seq 12,13 . Any glycan-binding probes, such as anti-glycan antibody and glycan-binding peptide, can be incorporated in Glycan-seq 32 . scGR-seq can be widely adapted to cells, organoids, and tissues and can be applied to various scientific fields such as stem cell biology, immunology, cancer biology, and neuroscience.…”

“…In terms of glycan-detection probes, cost-effective and commercially-available lectins, either from natural sources or recombinantly-expressed, were used for Glycan-seq 12,13 . Any glycan-binding probes, such as anti-glycan antibody and glycan-binding peptide, can be incorporated in Glycan-seq 32 .…”

“…scGR-seq could be readily adaptable to a droplet, micro-well, and indexing-based high-throughput singlecell technologies 9 . Any glycan-binding proteins such as anti-glycan antibodies and glycan-binding peptides could be incorporated in the developed system 16 . We believe that it has the potential to advance the understanding of the cellular heterogeneity as well as the biological roles of glycans across diverse multicellular systems in diverse species.…”

Detection of Human CD38 Using Variable Lymphocyte Receptor (VLR) Tetramers

“…The non-conventional VLR antibodies of the evolutionarily distant jawless vertebrates provide a source of anticipatory receptors with structural characteristics distinct from those of conventional mammalian immunoglobulins. Monoclonal VLR antibodies with specificities to carbohydrate antigens [18,[29][30][31][32] or tyrosine sulfation-dependent antigen recognition [21] emphasize the potential of VLR antibodies as a novel class of reagents in biomedical research applications. Previously, we isolated the decameric VLRB MM3 antibody whose recognition of human CD38 correlated with dimerization and CD38 enzymatic activity [19].…”