Glycoproteomic and glycomic databases

Protein glycosylation serves critical roles in the cellular and biological processes of many organisms. Aberrant glycosylation has been associated with many illnesses such as hereditary and chronic diseases like cancer, cardiovascular diseases, neurological disorders, and immunological disorders. Emerging mass spectrometry (MS) technologies that enable the high-throughput identification of glycoproteins and glycans have accelerated the analysis and made possible the creation of dynamic and expanding databases. Although glycosylation-related databases have been established by many laboratories and institutions, they are not yet widely known in the community. Our study reviews 15 different publicly available databases and identifies their key elements so that users can identify the most applicable platform for their analytical needs. These databases include biological information on the experimentally identified glycans and glycopeptides from various cells and organisms such as human, rat, mouse, fly and zebrafish. The features of these databases - 7 for glycoproteomic data, 6 for glycomic data, and 2 for glycan binding proteins are summarized including the enrichment techniques that are used for glycoproteome and glycan identification. Furthermore databases such as Unipep, GlycoFly, GlycoFish recently established by our group are introduced. The unique features of each database, such as the analytical methods used and bioinformatical tools available are summarized. This information will be a valuable resource for the glycobiology community as it presents the analytical methods and glycosylation related databases together in one compendium. It will also represent a step towards the desired long term goal of integrating the different databases of glycosylation in order to characterize and categorize glycoproteins and glycans better for biomedical research

Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion

In mammalian cells, >25% of synthesized proteins are exported through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important for biopharmaceutical production. Here we delineate the core secretory pathway functions and integrate them with genome-scale metabolic reconstructions of human, mouse, and Chinese hamster ovary\ua0cells. The resulting reconstructions enable the computation of energetic costs and machinery demands of each secreted protein. By integrating additional omics data, we find that highly secretory cells have adapted to reduce expression and secretion of other expensive host cell proteins. Furthermore, we predict metabolic costs and maximum productivities of biotherapeutic proteins and identify protein features that most significantly impact protein secretion. Finally, the model successfully predicts the increase in secretion of a monoclonal antibody after silencing a highly expressed selection marker. This work represents a knowledgebase of the mammalian secretory pathway that serves as a novel tool for systems biotechnology

N-linked glycosylation of Kv1.2 voltage-gated potassium channel facilitates cell surface expression and enhances the stability of internalized channels

KEY POINTS: Kv1.2 and related voltage‐gated potassium channels have a highly conserved N‐linked glycosylation site in the first extracellular loop, with complex glycosylation in COS‐7 cells similar to endogenous Kv1.2 glycosylation in hippocampal neurons. COS‐7 cells expressing Kv1.2 show a crucial role of this N‐linked glycosylation in the forward trafficking of Kv1.2 to the cell membrane. Although both wild‐type and non‐glycosylated mutant Kv1.2 channels that have reached the cell membrane are internalized at a comparable rate, mutant channels are degraded at a faster rate. Treatment of wild‐type Kv1.2 channels on the cell surface with glycosidase to remove sialic acids also results in the faster degradation of internalized channels. Glycosylation of Kv1.2 is important with respect to facilitating trafficking to the cell membrane and enhancing the stability of channels that have reached the cell membrane. ABSTRACT: Studies in cultured hippocampal neurons and the COS‐7 cell line demonstrate important roles for N‐linked glycosylation of Kv1.2 channels in forward trafficking and protein degradation. Kv1.2 channels can contain complex N‐linked glycans, which facilitate cell surface expression of the channels. Additionally, the protein stability of cell surface‐expressed Kv1.2 channels is affected by glycosylation via differences in the degradation of internalized channels. The present study reveals the importance of N‐linked complex glycosylation in boosting Kv1.2 channel density. Notably, sialic acids at the terminal sugar branches play an important role in dampening the degradation of Kv1.2 internalized from the cell membrane to promote its stability

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production