Dataset Information

Mass spectrometry analysis of the human endosulfatase Hsulf-2.

ABSTRACT: The human 6-O-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-O-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its functional properties remain poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133,115?Da was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98,170?Da), highlighting a significant contribution of post-translational modifications. The HSulf-2 protein sequence was determined by Nano-LC-MS/MS, leading to 63% coverage and indicating at least four N-glycosylation sites at Asn 108, 147, 174 and 217. These results provide a platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role of each chain in the substrate binding and specificities and in the catalytic activities.

SUBMITTER: Seffouh I

PROVIDER: S-EPMC6369371 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mass spectrometry analysis of the human endosulfatase Hsulf-2.

Seffouh Ilham I Przybylski Cédric C Seffouh Amal A El Masri Rana R Vivès Romain R RR Gonnet Florence F Daniel Régis R

Biochemistry and biophysics reports 20190207

The human 6-<i>O</i>-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-<i>O</i>-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its ...[more]

PMID: 30788440

Similar Datasets

Project description:Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05-0.50 versus 0.025-0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic information.

Dataset Information

Mass spectrometry analysis of the human endosulfatase Hsulf-2.

Publications

Mass spectrometry analysis of the human endosulfatase Hsulf-2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets