Glycan macro-and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro-and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3⌬ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation. Protein glycosylation is a highly conserved co-and posttranslocational modification of proteins that influences protein folding, stability, solubility, and function (1, 2). N-glycosylation of Asparagine (Asn) 1 residues occurs in eukaryota, archaea, and some bacteria, although the biosynthetic pathways and glycan structures are diverse in these organisms (3). In eukaryotes, nascent polypeptides in the Endoplasmic Reticulum (ER) are the protein acceptor substrates for N-glycosylation, as folded proteins cannot be efficiently N-glycosylated and N-glycosylation is critical for efficient protein folding. After protein folding, glycan structures can be further truncated or extended by Golgi-resident glycosyltransferases. Glycan biosynthesis is inherently inefficient, resulting in structural diversity of mature glycoproteins. Diversity in the presence or absence of glycans on glycoproteins is termed macroheterogeneity, whereas diversity in the structures of glycans at a specific site is termed microheterogeneity. This structural diversity is key for the regulation of many biological functions of glycoproteins...

Candida albicans is the most prevalent fungal pathogen of humans. The current techniques used to construct C. albicans strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays. We have identified a large intergenic region, NEUT5L, which facilitates the integration and expression of ectopic genes. To construct and integrate inserts into this novel locus, we re-engineered yeast/bacterial shuttle vectors by incorporating 550 bp of homology to NEUT5L. These vectors allow rapid, facile cloning through in vivo recombination (gap repair) in Saccharomyces cerevisiae and efficient integration of the construct into the NEUT5L locus. Other useful features of these vectors include a choice of three selectable markers (URA3, the recyclable URA3-dpl200 or NAT1), and rare restriction enzyme recognition sites for releasing the insert from the vector prior to transformation into C. albicans, thereby reducing the insert size and preventing integration of non-C. albicans DNA. Importantly, unlike the commonly used RPS1/RP10 locus, integration at NEUT5L has no negative effect on growth rates and allows native-locus expression levels, making it an ideal genomic locus for the integration of exogenous DNA in C. albicans.

Glycosylation is a co- and post-translational modification that is critical for the regulation of the biophysical properties and biological activities of diverse proteins. Biosynthetic pathways for protein glycosylation are inherently inefficient, resulting in high structural diversity in mature glycoproteins. Macroheterogeneity is the structural diversity due to the presence or absence of glycans at specific glycosylation sites, and is caused by inefficiency in the initial transfer of glycans to proteins. Here, we review the enzymatic and evolutionary mechanisms controlling macroheterogeneity, its biological consequences in physiological and disease states, its relevance to heterologous production and glycoengineering of glycoproteins, and mass spectrometry based methods for its analysis. We highlight the importance of the analysis of macroheterogeneity for a complete understanding of glycoprotein biosynthesis and function, and emphasize how advances in mass spectrometry glycoproteomics will enable analysis of this critical facet of glycoprotein structural diversity.