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Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.

In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD + ) under the hypothesis that NAD + regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD + treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD + precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD + biosynthesis was verified, and then the effect of NAD + precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD + levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD + treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD + treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD + precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD + precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD + precursors as medium and feed components for the biopharmaceutical production., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)