Generation and characterization of a series of monoclonal antibodies that specifically recognize [HexA(+/-2S)-GlcNAc]n epitopes in heparan sulfate.

Five monoclonal antibodies AS17, 22, 25, 38 and 48, a single monoclonal antibody ACH55, and three monoclonal antibodies NAH33, 43, 46, that recognize acharan sulfate (IdoA2S-GlcNAc)n, acharan (IdoA-GlcNAc)n and N-acetyl-heparosan (GlcA-GlcNAc)n, respectively, were generated by immunization of mice with keyhole limpet hemocyanin-conjugated polysaccharides. Specificity tests were performed using a panel of biotinylated GAGs that included chemically modified heparins. Each antibody bound avidly to the immunized polysaccharide, but did not bind to chondroitin sulfates, keratan sulfate, chondroitin nor hyaluronic acid.

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS.

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3..

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

Purpose: To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas.

Methods: Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Controlled release of fibroblast growth factor-2 from an injectable 6-O-desulfated heparin hydrogel and subsequent effect on in vivo vascularization.

We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding growth factors, such as fibroblast growth factor (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo.

Effects of heparin and its 6-O-and 2-O-desulfated derivatives with low anticoagulant activity on proliferation of human neural stem/progenitor cells.

Heparin binds various growth factors and activates them to interact with high-affinity cell surface receptors; a specific array of sulfate groups in the heparin backbone structure is very important for this interaction. In the present study, we evaluated the effects of two novel heparin derivatives, 6-O-desulfated heparin (6-DSH) and 2-O-desulfated heparin (2-DSH), on blood coagulation and the proliferation of human neural stem/progenitor cells (NSPCs). 6-DSH showed lower anticoagulant activity than intact heparin or 2-DSH, as measured by the activated partial thromboplastin time and thrombin time.

Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins.

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H.

Heparin structures in FGF-2-dependent morphological transformation of astrocytes.

Fibroblast growth factor-2 (FGF-2) participates in the morphological transformation of astrocytes (stellation) during the formation of glial scars in injured brains. In the current study, we used quantitative morphometric analysis to investigate the structural requirements for heparin's enhancement of FGF-2-induced stellation of cultured cortical astrocytes. Native heparin significantly promoted FGF-2-dependent astrocytic stellation, whereas heparin hexasaccharide inhibited FGF-2-dependent stellation.

Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS.

Purpose: To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Methods: Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4- and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes.

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry.

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS.

Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis.

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.

Attenuation of glial scar formation in the injured rat brain by heparin oligosaccharides.

Injury to the central nervous system causes glial reactions, which eventually lead to the formation of a glial scar and inhibit axonal regeneration. The present study aimed to reduce the extent of glial scar formation in injured cerebral cortex using heparin hexasaccharide (6-mer) and octasaccharide (8-mer). A single injection of 20 microl of heparin 6-mer or heparin 8-mer (10mg/ml), native heparin (10mg/ml), or saline vehicle was given into the wound cavity just after cryo-injury in the cerebral cortex.

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library.

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin.

Enhancement of neurite outgrowth-promoting activity by heparin derivatives in sodium chlorate-treated explant cultures of rat central neurons.

Periodate-oxidized/borohydride-reduced 2-O-desulfated heparin (OR2DSH) was prepared using intact heparin from pig intestine as the starting material. Successive treatments of the heparin by oxidation with sodium periodate and reduction with sodium borohydride yielded periodate-oxidized/borohydride-reduced heparin (OR-heparin). Subsequent 2-O-desulfation of OR-heparin, according to a previously established method, yielded OR2DSH.

Modification of di- and tetrasaccharides from shark cartilage keratan sulphate by refined anhydromethanolic hydrochloric acid-treatments and evaluation of their specific desulphation.

Highly sulphated keratan di- and tetrasaccharides were prepared from keratan sulphate (KS) of shark cartilage by enzymatic digestion with keratanase II and subsequent chromatography. The tetrasaccharide fraction carrying four sulphate groups was completely desulphated by 100 mM anhydromethanolic hydrochloric acid (MeOH-HCl) treatment at room temperature for 16 h. The conditions for the desulphation reaction by MeOH-HCl treatment were examined using sulphated keratan di- and tetrasaccharides as substrates by means of reversed phase high performance liquid chromatography (HPLC) and/or capillary electrophoresis, followed by the preparation of partially desulphated keratan oligosaccharides.

Enhancement of t-PA-mediated plasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicus.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives.