Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides

Vivès, Romain R.; Goodger, Sarah J.; Pye, David

doi:10.1042/bj3540141

Cited by 18 publications

(9 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PAGE of HS Fragments-The HS fragments were analyzed by PAGE to determine the level of purity of each one according to a previously described method (29). Each HS fragment (5 g) was mixed with 20% glycerol up to a maximum volume of 10 -15 l and then loaded into separate wells (Fig.…”

Section: Methodsmentioning

confidence: 99%

The Solution Structure of Heparan Sulfate Differs from That of Heparin

Khan

Rodríguez

Patel

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments degree of polymerization 6 -18 (dp6 -dp18) and dp24, corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s 20,w of HS dp6 -dp24 showed a small rotor speed dependence, where similar s 20,w values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6 -dp24 gave radius of gyration (R G ) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (R XS ) values from 0.31 to 0.65 nm, and maximum lengths (L) from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5000 -8000 conformationally randomized HS structures gave best fit dp6 -dp16 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dp18 or dp24, indicating their higher flexibility. We conclude that HS displays an extended bent conformation that is significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.

show abstract

Section: Methodsmentioning

confidence: 99%

The Solution Structure of Heparan Sulfate Differs from That of Heparin

Khan

Rodríguez

Patel

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…TNF-␣-based fluorescent peptide substrate (dabcyl-LAQAhomoPheRSK(5FAM)-NH 2 ) was from BioZyme, Inc. Heparin beads (TSK gel heparin 5PW) were from Tosoh Bioscience. Heparin oligosaccharides were prepared as described (29). Heparin, heparan sulfate, dermatan sulfate, L-chondroitin 4-and 6-sulfate, completely desulfated heparin, and IGF-I were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Heparan Sulfate Regulates ADAM12 through a Molecular Switch Mechanism

Sørensen

Vivès

Manetopoulos

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.

show abstract

“…A conventional SAX method using a Propac PA1 column was tested with volatile ammonium bicarbonate salts; however, neither disaccharides nor oligosaccharides eluted from the column, indicating the interaction was too strong for ammonium bicarbonate to achieve effective dissociation (data not shown). To overcome this, we investigated CTA-SAX in which a C-18 reverse phase column is first derivatized using CTA, imparting a positive charge on the matrix and allowing the column to be used for SAX. First, eight commonly occurring HS disaccharide standards were separated on CTA-SAX using a 0–1 M ammonium methanesulfonic acid gradient over 60 min (Figure a).…”

Section: Resultsmentioning

confidence: 99%

“…SAX is a powerful heparin/HS separation method, as it offers higher oligosaccharide resolution over other methods currently available, including hydrophilic interaction chromatography (HILIC), reverse phase ion pairing (RP-IP), , and capillary electrophoresis (CE). , With small saccharides (for example, disaccharides), there is a significant degree of separation dependent on the presence of 2OS, 6OS, and NS groups, but as the oligosaccharide becomes longer, this resolution decreases, necessitating the use of additional SAX or orthogonal chromatography methods.…”

mentioning

confidence: 99%

Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography

et al. 2016

View full text Add to dashboard Cite

The complexity of heparin and heparan sulfate saccharides makes their purification, including many isomeric structures, very challenging and is a bottleneck for structure-activity studies. High-resolution separations have been achieved by strong anion exchange (SAX) chromatography on Propac PA1 and cetyltrimethylammonium (CTA)-C silica columns; however, these entail subsequent desalting methodologies and consequent sample losses and are incompatible with orthogonal chromatography methodologies and, in particular, mass spectrometry. Here, we present the CTA-SAX purification of heparin oligosaccharides using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium bicarbonate buffer for elution improves resolution through both weaker dissociation and conformational coordination of the ammonium across the sulfate groups. Using ion mobility mass spectrometry, we demonstrate that isomeric structures have different structural conformations, which makes chromatographic separation achievable. Resolution of such structures is improved compared to standard SAX methods, and in addition, VSCTA-SAX provides an orthogonal method to isolate saccharides with higher purity. Because ammonium bicarbonate is used, the samples can be evaporated rather than desalted, preventing substantial sample loss and allowing more effective subsequent analysis by electrospray mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool that helps address the difficult challenge of heparin/heparan sulfate saccharide separation and will enhance structure-activity studies.

show abstract

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides

Cited by 18 publications

References 22 publications

The Solution Structure of Heparan Sulfate Differs from That of Heparin

The Solution Structure of Heparan Sulfate Differs from That of Heparin

Heparan Sulfate Regulates ADAM12 through a Molecular Switch Mechanism

Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography

Contact Info

Product

Resources

About