This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The recently discovered Sulf2 enzyme acts to modify heparan sulfate (HS) associated with cell surface and extracellular matrix proteoglycans. This remodeling affects cellular recognition of growth factor families including transforming growth factors, vascular endothelial growth factors, and fibroblast growth factors. Detailed structural information on the HS domain context required for Sulf2 activity is lacking due to analytical challenges. Such information is essential to understanding the functional roles of Sulf2 enzymes as a function of tissue location and temporal changes in the growth environment (development and disease). Over the past year we have use d the LC/MS platform to profile changes to HS structure mediated by recombinant Sulf2 enzyme. Data were collected by digesting HS from different bovine organs with Sulf2 and then treating with lyase enzymes. Exhaustive depolymerization followed by LC/MS extended the information on the disaccharide sites recognized by Sulf enzymes to include 2 sites not previously identified. This work was published recently (1). In continuing work, porcine intestine mucosa HS was partially digested by heparin lyases I and III. HS oligosaccharides of degree of polymerization of 6-8 (dp6-8) were fractionated by SEC and collected. Part of the faction was treated with recombinant HSulf2 provided by Shire Pharmaceuticals. An equal quantity was treated with digestion buffer only as control. Both HSulf2 treated and control dp6-8 fractions were analyzed by a custom packed hydrophilic interaction chromatography HPLC-chip with pulsed make-up flow online with an Agilent 6520 QTOF. Those oligosaccharides exhibiting pronounced change with HSulf2 treatment were subjected to LC-MS/MS, with sulfolane pulsed at the corresponding retention time to elevate their charge states for better tandem MS. Sulfs act on a subset of 6O-sulfates within HS chains. Our previously study of HSulf2 treatment followed by exhaustive heparin lyase depolymerization has shown that HSulf2 cleaves 6O-sulfate in the non-reducing end as well. However, due to the lack of structurally defined HS oligosaccharides, the elucidation of structural specificity of HSulf2 remains a challenge. In this study, we used oligosaccharides with reasonable lengths, of which isomeric profiles were simplified by lyase specificity, to discern the substrate structural preferences of HSulf2. HSulf2 acts preferentially on highly sulfated dp6-8 with 0-1 acetate group. At the same time, the abundance of relatively low sulfated dp6-8 increased as the products by HSulf2. The average sulfation degree decreased more for dp6-8 with zero acetate than for those with one acetate group. Although dp6-8 with 2 acetate groups were highly sulfated and contain 6O-sulfation, HSulf2 did not change the profiles of dp6-8 with 2 acetate groups, suggesting its preference for few acetates and highly sulfated oligosaccharides. Since only 6O-sulfation is cleaved by HSulf2, only certain isomers were affected and this was reflected by the chromatographic peak shape change of the extracted ion chromatograms (EICs). Those dp6-8 that were suspected to experience these changes were chosen as targets for tandem MS. A customized pulsed make-up flow HPLC-chip was used to pulsed sulfolane during the elution time of the target precursors to enhance their charge states for more informative tandem mass spectra. Although these tandem mass spectra seem to look similar for entire EIC peak range and entire collision energy range, by careful selection of retention time windows and collision energy, the spectra differences were made more evident, suggesting a subset of the oligosaccharides have been processed and HSulf2. These tandem mass spectra have the potential to provide more structural information of the substrate specificity of HSulf2. 1. Staples, G. O., Shi, X., and Zaia, J. (2011) Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2, PLoS ONE 6, e16689.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-15
Application #
8365551
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Project Start
2011-06-01
Project End
2012-08-09
Budget Start
2011-06-01
Budget End
2012-08-31
Support Year
15
Fiscal Year
2011
Total Cost
$12,304
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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