STRUCTURAL ANALYSIS OF SULF2 PROCESSING OF EXTRACELLULAR HEPARAN SULFATE

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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). Zaia et al have developed a liquid chromatography-mass spectrometric (LC/MS) platform for analysis of heparin sulfates and other glycosaminoglycan classes. This platform includes hydrophilic interaction chromatography and high resolution electrospray mass spectrometry. A tetraplex glycan stable isotope tag is used to facilitate quantification of HS oligosaccharides. This platform is ideal for assessing the activity of Sulf2 enzymes. Sulf2 activity will be tested versus HS derived from several organs available at the MSR. HS samples will be digested with Sulf2. Samples will then be partially depolymerized using heparin lyases so as to produce oligosaccharides ranging from dimers to decamers. The oligosaccharides will be derivatized using tetraplex glycan tags to facilitate quantification. The oligosaccharide compositions will be determined by high accuracy mass measurement using an LC-LTQ-Orbitrap MS system. Oligosaccharides observed to undergo changes in abundance will be analyzed using tandem mass spectrometry. The product ion pattern will be used to determine HS oligosaccharide structures. It is expected that accurate mass measurement will define the HS domain (N-sulfated, N-acetylated, or hybrid) from which the oligosaccharide derives. Changes in abundance will determine the overall domain preference of Sulf2 as a function of mammalian organ. Tandem MS will provide additional structural detail regarding the sites acted upon by Sulf2.