By-Products of Heparin Production Provide a Diverse Source of Heparin-like and Heparan Sulfate Glycosaminoglycans

Abstract

Global production of pharmaceutical heparin (Hp) is increasing, and the production process from raw mucosal material results in large amounts of waste by-products. These contain lower sulfated Hp-like and heparan sulfate (HS), as well as other glycosaminoglycans, which are bioactive entities with pharmaceutical potential. Here we describe the first purification, structural and functional characterisation of Hp-like and HS polysaccharides from the four major by-product fractions of standard heparin production. Analysis of the by-products by disaccharide composition analysis and NMR demonstrated a range of structural characteristics which differentiate them from Hp (particularly reduced sulfation and sulfated disaccharide content), and that they are each distinct. Functional properties of the purified by-products varied, each displaying distinct anticoagulant profiles in different assays, and all exhibiting significantly lower global and specific inhibition of the coagulation pathway than Hp. The by-products retained the ability to promote cell proliferation via fibroblast growth factor receptor signalling, with only minor differences between them. These collective analyses indicate that they represent an untapped and economical source of structurally-diverse Hp-like and HS polysaccharides with the potential for enhancing future structure-activity studies and uncovering new biomedical applications of these important natural products.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides

“…The nonasaccharide #26 lacks 6OS and yet partially promotes FGF2 signaling, demonstrating the utility of complete characterization of sequences by SIMMS 2 in deciphering cues for FGF bioactivities. We then obtained HS byproducts from pharmaceutical heparin production 42 , and used limited heparinase III digestion to produce a range of oligosaccharide size fractions, which were initially separated by size exclusion chromatography (SEC). The dp8 and dp6 fractions were further separated through strong anion exchange (SAX)-HPLC and selected fractions were tested for bioactivity in the BaF3 assay (See Supplementary Fig.…”

“…BaF3 FGF signaling assays. The activity of HS oligosaccharides with FGF1 and FGF2 was determined using BaF3 cells transfected with FGFR1c (from David Ornitz, Washington University, St. Louis) 39,42 . BaF3 cells (1 × 10 4 cells/well) were plated into 96 well plates with recombinant FGF-1 or FGF-2 (1 ng/mL; R&D Systems) and either heparin or oligosaccharides (3 µg/mL).…”

Hydrolytic Degradation of Heparin in Acidic Environments: Nuclear Magnetic Resonance Reveals Details of Selective Desulfation

“…The previous studies confirmed that loss of 6-O-sulfate is associated with the major chemical shift of A-6 and A-5 carbon signals. 47,52 The 13 C spectra comparison of heparin standard and pH 1/ 80 C, indeed, revealed the gradual upfield changes from 69.1 to 62.3 ppm (6.9 ppm) at positions A-6 and downfield move of the A-5 signal, from 71.9 to 74.2 ppm (2.3 ppm). The proton positions of both peaks moved upfield by 0.03 and 0.06 ppm, (Table 3).…”

Steroid disulfates - Sulfation double trouble

“…One example is the N-terminus of the cytokine receptor protein CCR5; it needs to be sulfated twice to serve as anchor point for its natural ligand CCL5 and for HIV (Abayev, Rodrigues, Srivastava et al, 2018). Another example is the widely used anti-coagulation medication heparin, a glycosaminoglycan which is known as one of the most highly sulfated biomolecule per weight (Taylor, Hogwood, Guo et al, 2019).…”