Heparanase as an Additional Tool for Detecting Structural Peculiarities of Heparin Oligosaccharides

Abstract

Due to the biological properties of heparin and low-molecular-weight heparin (LMWH), continuous advances in elucidation of their microheterogeneous structure and discovery of novel structural peculiarities are crucial. Effective strategies for monitoring manufacturing processes and assessment of more restrictive specifications, as imposed by the current regulatory agencies, need to be developed. Hereby, we apply an efficient heparanase-based strategy to assert the structure of two major isomeric octasaccharides of dalteparin and investigate the tetrasaccharides arising from antithrombin binding region (ATBR) of bovine mucosal heparin. Heparanase, especially when combined with other sample preparation methods (e.g., size exclusion, affinity chromatography, heparinase depolymerization), was shown to be a powerful tool providing relevant information about heparin structural peculiarities. The applied approach provided direct evidence that oligomers bearing glucuronic acid–glucosamine-3-O-sulfate at their nonreducing end represent an important structural signature of dalteparin. When extended to ATBR-related tetramers of bovine heparin, the heparanase-based approach allowed for elucidation of the structure of minor sequences that have not been reported yet. The obtained results are of high importance in the view of the growing interest of regulatory agencies and manufacturers in the development of low-molecular-weight heparin generics as well as bovine heparin as alternative source.

Heparinase Digestion of 3-O-Sulfated Sequences: Selective Heparinase II Digestion for Separation and Identification of Binding Sequences Present in ATIII Affinity Fractions of Bovine Intestinal Heparins

“…Structures tentatively proposed were ΔIIs- IVs glu [ΔHexUA-GlcN(NS,6S)-GlcA-GlcN(NS,3S)] and ΔIIIs- IVs glu [ΔHexUA(2S)-GlcNS-GlcA-GlcN(NS,3S)] for the two at 994 Da, and ΔIIIs- IIs glu and ΔIs- IVs glu for those at 1074 Da. In accordance with Naggi et al ( 27 , 44 ), ΔIIIs- IIs glu and ΔIs- IVs glu appear to be appropriate choices for two of the five ΔU(4,5,0) unknowns in light of their distribution in the affinity fractions and their transformation by Δ 4−5 glycuronidase and Δ 4−5 2- O -sulfatase.…”

“…Low amounts (<0.1%) of the ΔU(4,3,0) tetrasaccharide ΔIVs- IVs glu [ΔHexUA-GlcNS-GlcA-GlcN(NS,3S)] (initially present in trace amounts) were also obtained when Δ 4−5 2- O -sulfatase was added. This was due to digestion of ΔIIIs- IVs glu as proposed in ( 44 ) as a possible ΔU(4,4,0). However, additionally, peaks ΔU(4,4,0)-2 and ΔU(4,4,0)-3 increased as a result of the enzymatic Δ 4−5 2- O -desulfation of ΔIIIs- IIs glu and ΔIs- IVs glu .…”

“…The constituents of exhaustive digests from BMH ATIII affinity fractions have already been described by Naggi et al ( 27 , 44 ). Four supplementary 3S tetrasaccharide building blocks (two with molecular weight Mw 994 Da [ΔU(4,4,0)] and two at 1074 Da [ΔU(4,5,0)] were detected in this work.…”

Prevention of Herpesviridae Infections by Cationic PEGylated Carbosilane Dendrimers

“…In additional experiments, cells were incubated for 1 h at 37 °C with dendrimers at the same concentration and washed with PBS containing 2.0 M NaCl, known to disrupt the binding to HSPGs [ 31 ]. Along with these experiments, cells were pre-treated with 200 mU/mL of heparinase II, acknowledged to inhibit HSPG-dependent binding [ 32 ], (Merck Group, Darmstadt, Germany) for 2 h at 37 °C, before incubating for 1 h at 37 °C with dendrimers at this concentration. Assessment of the amounts of cell-associated dendrimers after these studies was done by evaluating the mean fluorescence intensity (MFI) of dendrimers in cells by flow cytometry, as described in its corresponding section.…”

The Auxiliary Role of Heparin in Bone Regeneration and its Application in Bone Substitute Materials

“…The inhibitory effects of heparin and AT vary and depends on the affinity of heparin with AT which can be variable. The antithrombin-binding region of heparin was identified as a pentasaccharide sequence, with the structure GlcNAc6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S, with the 3-O-sulfate and 6-O-sulfate structures involved specifically in AT binding ( Alekseeva et al, 2019 ). In summary, heparin exists as a linear, unbranched, and deeply sulfated GAG, which primarily consists of the helical structure ( Mulloy et al, 1993 ).…”