{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T09:05:19Z","timestamp":1784019919780,"version":"3.55.0"},"reference-count":35,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Heparin, the naturally occurring, highly sulfated glycosaminoglycan drug, acts as a regulator of diverse biological processes by interacting with diverse specific proteins through a variety of sulfated structural motifs with varying degrees of affinity. Despite its rarity, 3-O-sulfation of glucosamine plays a key role in several biological activities, especially anticoagulant activity mediated by interaction with antithrombin (AT), a mechanism that has been extensively studied. The present work primarily focuses on sequences containing 3-O-sulfated glucosamine that are not involved in anticoagulant activity, analysing their relative abundance and structural environments in heparins derived from distinct animal origins. Three heparin samples derived from bovine, ovine and porcine intestinal mucosa (BMH, OMH and PMH) were fractionated by affinity chromatography on AT-Sepharose into no affinity (NA) and high affinity (HA) fractions. Parent heparins and derived NA and HA fractions were enzymatically depolymerised using either a cocktail of heparinases I, II and III or heparinase II alone, and the resulting mixtures of di- and oligosaccharides were analysed by liquid chromatography coupled with mass spectrometry. Digestion with heparinase II, which preserves heparin sequences containing 3-O-sulfated glucosamine, produced a series of 3-O-sulfated trisaccharides containing two glucosamine residues either side of a uronic acid, located at the non-reducing end (NRE) of heparin chains. Treatment with a heparinase cocktail, which cleaves these trisaccharides, released NRE glucosamine monosaccharides, including 3-O-sulfated species. Notably, both the number of NRE trisaccharide species and the overall proportion of 3-O-sulfated monosaccharides were markedly higher in OMH compared to BMH or PMH. Using 1H\/13C bi-dimensional nuclear magnetic resonance spectroscopy, the higher proportion of NRE 3-O-sulfated glucosamine in ovine heparin relative to bovine and porcine heparins was found to be preferentially located in chains lacking affinity for AT. These results offer new insights into the localisation of most 3-O-sulfated heparin sequences that are not associated with anticoagulant activity and, importantly, also reveal a possible structural marker that indicates the ovine origin of heparin. Additionally, a tetrasaccharide species was detected that may be indicative of a highly sulfated AT-binding site in BMH.<\/jats:p>","DOI":"10.3390\/biom16071025","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T08:04:31Z","timestamp":1784016271000},"page":"1025","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Content and Location of 3-O-Sulfated Glucosamine Enable Differentiation of Ovine, Bovine and Porcine Heparins"],"prefix":"10.3390","volume":"16","author":[{"given":"Arianna","family":"Somma","sequence":"first","affiliation":[{"name":"Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0918-0808","authenticated-orcid":false,"given":"Elena","family":"Urso","sequence":"additional","affiliation":[{"name":"Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5538-5978","authenticated-orcid":false,"given":"Michela","family":"Parafioriti","sequence":"additional","affiliation":[{"name":"Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Guerrini","sequence":"additional","affiliation":[{"name":"Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonella","family":"Bisio","sequence":"additional","affiliation":[{"name":"Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.2741\/4462","article-title":"Heparin and Anticoagulation","volume":"21","author":"Onishi","year":"2016","journal-title":"Front. Biosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1177\/10760296231173965","article-title":"Blended Heparin: A New Perspective to Guarantee the Supply of a Life-Saving Drug?","volume":"29","author":"Guerrini","year":"2023","journal-title":"Clin. Appl. Thromb.\/Hemost."},{"key":"ref_3","first-page":"1","article-title":"General Considerations for Diversifying Heparin Drug Products by Improving the Current Heparin Manufacturing Process and Reintroducing Bovine Sourced Heparin to the US Market","volume":"27","year":"2021","journal-title":"Clin. Appl. Thromb. Hemost."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Villanova, E., Vairo, B.C., Oliveira, S.-N.M.C.G., Glauser, B.F., Capill\u00e9, N.V., Santos, G.R.C., Tovar, A.M.F., Pereira, M.S., and Mour\u00e3o, P.A.S. (2019). Heparins Sourced from Bovine and Porcine Mucosa Gain Exclusive Monographs in the Brazilian Pharmacopeia. Front. Med., 6.","DOI":"10.3389\/fmed.2019.00016"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1016\/j.ijcard.2016.08.033","article-title":"A Brazilian perspective foe the use of bovine heparin in open heart surgery","volume":"223","author":"Gomes","year":"2016","journal-title":"Int. J. Cardiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5027","DOI":"10.1182\/blood.V128.22.5027.5027","article-title":"Porcine and Ovine Mucosal Heparins and Their Depolymerized Derivatives Are Comparable in Contrast to Their Bovine Equivalent","volume":"128","author":"Bouchard","year":"2016","journal-title":"Blood"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1177\/1076029619889406","article-title":"Comparative pharmacological profiles of various bovine, ovine and porcine heparins","volume":"25","author":"Kouta","year":"2019","journal-title":"Clin. Appl. Thromb. Hemost."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Jeske, W., Kouta, A., Farooqui, A., Siddiqui, F., Rangnekar, V., Niverthi, M., Laddu, R., Hoppensteadt, D., Iqbal, O., and Walenga, J. (2019). Bovine mucosal heparins are comparable to porcine mucosal heparin at USP potency adjusted levels. Front. Med., 5.","DOI":"10.3389\/fmed.2018.00360"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5577","DOI":"10.1182\/blood-2024-211016","article-title":"Unfractionated Heparin Resourced from Bovine, Ovine, and Porcine Origin Is Bioequivalent","volume":"144","author":"Thakkar","year":"2024","journal-title":"Blood"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1093\/glycob\/cwg006","article-title":"Heparin sequencing","volume":"13","author":"Stringer","year":"2003","journal-title":"Glycobiology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6551","DOI":"10.1073\/pnas.77.11.6551","article-title":"Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin","volume":"77","author":"Lindahl","year":"1980","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1042\/bj3590265","article-title":"Conformation of heparin pentasaccharide bound to antithrombin III","volume":"359","author":"Hricovini","year":"2001","journal-title":"Biochem. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1016\/S0021-9258(19)57387-2","article-title":"Location of specific oligosaccharides in heparin in terms of their distance from the protein linkage region in the native proteoglycan","volume":"263","author":"Rosenfeld","year":"1988","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1863","DOI":"10.1074\/jbc.273.4.1863","article-title":"A major common trisulfated hexasaccharide core sequence, hexuronic acid(2-sulfate)-glucosamine(N-sulfate)-iduronic acid-N-acetylglucosamine-glucuronic sequence, acid-glucosamine(N-sulfate), isolated from the low sulfated irregular region of porcine intestinal heparin","volume":"273","author":"Yamada","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/S0021-9258(17)43636-2","article-title":"Location on heparin of the oligosaccharide section essential for anticoagulant activity","volume":"259","author":"Radoff","year":"1984","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"35152","DOI":"10.1074\/jbc.M300925200","article-title":"Processing of macromolecular heparin by heparinase","volume":"278","author":"Gong","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.matbio.2013.12.001","article-title":"Heparan sulfate 3-O-sulfation: A rare modification in search of a function","volume":"35","author":"Thacker","year":"2014","journal-title":"Matrix Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1447","DOI":"10.1002\/jps.23501","article-title":"Structural Characterization of Pharmaceutical Heparins Prepared from Different Animal Tissues","volume":"102","author":"Fu","year":"2013","journal-title":"J. Pharm. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Mourier, P. (2022). 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. Front. Med., 9.","DOI":"10.3389\/fmed.2022.841726"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Mourier, P. (2022). Building Block Analysis of ATIII Affinity Fractions of Heparins: Application to the ATIII Binding Capacity of NON-conventional 3-O-Sulfated Sequences. Front. Med., 9.","DOI":"10.3389\/fmed.2022.841738"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/0014-5793(76)80592-3","article-title":"Anticoagulant activity of heparin: Separation of high-activity and low-activity heparin species by affinity chromatography on immobilized antithrombin","volume":"66","author":"Hopwood","year":"1976","journal-title":"FEBS Lett."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1016\/0003-2697(62)90095-7","article-title":"A modified Uronic Acid Carbazole Reaction","volume":"4","author":"Bitter","year":"1962","journal-title":"Anal. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Alekseeva, A., Urso, E., Mazzini, G., and Naggi, A. (2019). Heparanase as an Additional Tool for Detecting Structural Peculiarities of Heparin Oligosaccharides. Molecules, 24.","DOI":"10.3390\/molecules24234403"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"108540","DOI":"10.1016\/j.carres.2022.108540","article-title":"Structural Variation in the Linkage Region of Pharmaceutical Heparin Arising from Oxidative Treatments during Manufacture","volume":"514","author":"Urso","year":"2022","journal-title":"Carbohydr. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.jpba.2016.12.031","article-title":"Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins","volume":"136","author":"Mauri","year":"2017","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Mourier, P.A.J. (2020). Specific Non-reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid. Molecules, 25.","DOI":"10.3390\/molecules25235553"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8140","DOI":"10.1021\/bi00083a012","article-title":"Specificity Studies on the Heparin Lyases from Flavobacterium heparinum","volume":"32","author":"Desai","year":"1993","journal-title":"Biochemistry"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Mauri, L., Marinozzi, M., Phatak, N., Karfunkle, M., St. Ange, K., Guerrini, M., Keire, D.A., and Linhardt, R.J. (2019). 1D and 2D-HSQC NMR: Two Methods to Distinguish and Characterize Heparin from Different Animal and Tissue Sources. Front. Med., 6.","DOI":"10.3389\/fmed.2019.00142"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"7487","DOI":"10.1021\/ac4014379","article-title":"Unravelling structural information from complex mixtures utilizing correlation spectroscopy applied to HSQC Spectra","volume":"85","author":"Rudd","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.jpba.2015.10.001","article-title":"Structural Peculiarity of Antithrombin Binding Region Profile of Mucosal Bovine and Porcine Heparins","volume":"118","author":"Naggi","year":"2016","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e2012935118","DOI":"10.1073\/pnas.2012935118","article-title":"The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation","volume":"118","author":"Chopra","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"15874","DOI":"10.1016\/S0021-9258(18)55480-6","article-title":"Characterization of novel sequences containing 3-O-sulfated glucosamine in glomerular basement membrane heparan sulfate and localization of sulfated disaccharides to a peripheral domain","volume":"265","author":"Edge","year":"1990","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5185","DOI":"10.1074\/jbc.274.8.5185","article-title":"Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities","volume":"274","author":"Liu","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1042\/BJ20121309","article-title":"An unusual antithrombin-binding octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence","volume":"449","author":"Guerrini","year":"2013","journal-title":"Biochem. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11814","DOI":"10.1002\/chem.202001346","article-title":"Degeneracy of the antithrombin binding sequence in heparin: 2-O-sulfated Iduronic acid can replace the critical glucuronic acid","volume":"26","author":"Elli","year":"2020","journal-title":"Chem. Eur. J."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/7\/1025\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T08:19:16Z","timestamp":1784017156000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/7\/1025"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,14]]},"references-count":35,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["biom16071025"],"URL":"https:\/\/doi.org\/10.3390\/biom16071025","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,14]]}}}