Human core 1 β3-galactosyltransferase (C1β3Gal-T) generates the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (T antigen), which is a precursor for many extended O-glycans in animal glycoproteins. We report here that C1β3Gal-T activity requires expression of a molecular chaperone designated Cosmc (core 1 β3-Gal-T-specificmolecularchaperone). The humanCosmcgene is X-linked (Xq23), and its cDNA predicts a 318-aa transmembrane protein (≈36.4 kDa) with type II membrane topology. The human lymphoblastoid T cell line Jurkat, which lacks C1β3Gal-T activity and expresses the Tn antigen GalNAcα1-Ser/Thr, contains a normal gene and mRNA encoding C1β3Gal-T, but contains a mutatedCosmcwith a deletion introducing a premature stop codon. Expression of Cosmc cDNA in Jurkat cells restored C1β3Gal-T activity and T antigen expression. Without Cosmc, the C1β3Gal-T is targeted to proteasomes. Expression of active C1β3Gal-T in Hi-5 insect cells requires coexpression of Cosmc. Overexpression of active C1β3Gal-T in mammalian cell lines also requires coexpression of Cosmc, indicating that endogenous Cosmc may be limiting. A small portion of C1β3Gal-T copurifies with Cosmc from cell extracts, demonstrating physical association of the proteins. These results indicate that Cosmc acts as a specific molecular chaperone in assisting the folding/stability of C1β3Gal-T. The identification of Cosmc, a uniquely specific molecular chaperone required for a glycosyltransferase expression in mammalian cells, may shed light on the molecular basis of acquired human diseases involving altered O-glycosylation, such as IgA nephropathy, Tn syndrome, Henoch-Schönlein purpura, and malignant transformation, all of which are associated with a deficiency of C1β3Gal-T activity.

Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving; however, the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use a combination of machine learning algorithms and expert annotation to define lectin specificity for this important probe set. Our analysis uses comprehensive glycan microarray analysis of commercially available lectins we obtained using version 5.0 of the Consortium for Functional Glycomics glycan microarray (CFGv5). This data set was made public in 2011. We report the creation of this data set and its use in large-scale evaluation of lectin–glycan binding behaviors. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides and linkages. Combining machine learning with manual annotation, we create a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.

The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza A virus are responsible for the surface interactions of the virion with the host. Entry of the virus is mediated by functions of the HA: binding to cellular receptors and facilitating fusion of the virion membrane with the endosomal membrane. The HA structure contains receptor binding sites in the globular membrane distal head domains of the trimer, and the fusion machinery resides in the stem region. These sites have specific characteristics associated with subtype and host, and the differences often define species barriers. For example, avian viruses preferentially recognize α2,3-Sialic acid terminating glycans as receptors and mammalian viruses recognize α2,6-Sialic acid. The neuraminidase, or the receptor-destroying protein, cleaves the sialic acid from cellular membrane constituents and viral glycoproteins allowing for egress of nascent virions. A functional balance of activity has been demonstrated between the two glycoproteins, resulting in an optimum level of HA affinity and NA enzymatic cleavage to allow for productive infection. As more is understood about both HA and NA, the relevance for functional balance between HA and NA continues to expand, with potential implications for interspecies transmission, host adaptation, and pathogenicity.

Treating glycoproteins with household bleach releases N- and O-glycans for further structural probing. Bleach treatment of glycosphingolipids releases glycan nitriles. Glycans have essential roles in biology and the etiology of many diseases. A major hurdle in studying glycans through functional glycomics is the lack of methods to release glycans from diverse types of biological samples. Here we describe an oxidative strategy using household bleach to release all types of free reducing N-glycans and O-glycan-acids from glycoproteins, and glycan nitriles from glycosphingolipids. Released glycans are directly useful in glycomic analyses and can be derivatized fluorescently for functional glycomics. This chemical method overcomes the limitations in glycan generation and promotes archiving and characterization of human and animal glycomes and their functions.