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. Introduction Squamous cell carcinoma (SCC) is one major cancer type that occurs in esophageal cancer, head and neck cancer. The invasiveness and progression of SCC are affected by the interactions between the malignant cells and the stroma around them. Stroma is characterized by collagen-rich extra-cellular matrix, as well as proteoglycans within. Proteoglycans consist of protein cores, attached with one or multiple chains of chondroitin sulfate and dermatan sulfate. These glycosaminoglycan chains bind with growth factors, mediating cell proliferation, adhesion, migration, and ultimately, cancer metastasis. In addition, the escalated activity of chondroitin sulfate C-5 epimerase has been observed in esophageal cancer. Therefore, there is a clear need to systematically characterize chondroitin sulfate and dermatan sulfate in cancerous biopsy samples. Method Biopsies of cancerous (containing both carcinoma and stroma) and normal areas of were surgically removed from esophageal cancer patients, and their epimerase activities were determined by enzymatic studies. The samples were digested by pronase and benzonase sequentially. The glycosaminoglycans were released by NaOH and recovered by weak anion exchange. GAGs were digested with chondroitinase ABC and chondroitinase B, respectively. The disaccharides and oligosaccharides were analyzed by SEC LC/MS, equipped with a Waters Acquity UPLC, a GE Bioscience Superdex peptide column, and an ABSciex Qstar mass spectrometer. The disaccharides'profiles were obtained by comparing the relative and absolute abundances using integration of EICs. The -4 or -6 position sulfate of the galactosamine was differentiated by LC/MS/MS. Results We successfully extracted and purified glycosaminoglycans (GAGs) from biopsy tissue samples and were able to digest and analyze chondroitin sulfate (CS) and dermatan sulfate (DS) in the presence of intact heparan sulfate. This GAG extraction and analysis platform is shown to be capable to analyze CS and DS disaccharides and oligosaccharides with high robustness and reproducibility. Some consistent patterns of CS/DS between cancerous and normal biopsy samples were observed. The cancerous samples have drastically increased expression of CS/DS, 60% to 400% higher than normal tissues of the same patient. In average, the cancerous samples also appear to have higher sulfation degree than normal biopsies. The singly sulfated disaccharides, as a result of lyase depolymerization, have different isomeric distribution: the percentage of 4-sulfated disaccharides appear to be slightly lower in cancerous samples than in normal samples. When the CS/DS is digested by chondroitinase B, which only acts on the iduronic acid-containing sites, both disaccharides of one sulfate and tetrasaccharide with two sulfates are observed as major depolymerized species. The singly-sulfated dp2 was analyzed by LC/MS/MS and was confirmed to be exclusively 4-sulfated, leading to the assignment of these disaccharides to originate from continuous DS chains or domains. The dp4's with two sulfates are likely from CS/DS domains with alternating iduronic acid and glucuronic acid containing disaccharide units. The disaccharides released by chondroitinase B digest compared with total disaccharides and oligosaccharides released by chondroitinase ABC, indicative of consecutive DS content in total CS/DS, appear to be lower in cancerous biopsy samples than normal ones. Therefore, there is an inverse relationship between the epimerase activity in biopsy samples and their DS content.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-15
Application #
8365534
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
$40,000
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Hu, Han; Khatri, Kshitij; Zaia, Joseph (2017) Algorithms and design strategies towards automated glycoproteomics analysis. Mass Spectrom Rev 36:475-498
Sethi, Manveen K; Zaia, Joseph (2017) Extracellular matrix proteomics in schizophrenia and Alzheimer's disease. Anal Bioanal Chem 409:379-394
Hu, Han; Khatri, Kshitij; Klein, Joshua et al. (2016) A review of methods for interpretation of glycopeptide tandem mass spectral data. Glycoconj J 33:285-96
Ji, Yuhuan; Bachschmid, Markus M; Costello, Catherine E et al. (2016) S- to N-Palmitoyl Transfer During Proteomic Sample Preparation. J Am Soc Mass Spectrom 27:677-85
Pu, Yi; Ridgeway, Mark E; Glaskin, Rebecca S et al. (2016) Separation and Identification of Isomeric Glycans by Selected Accumulation-Trapped Ion Mobility Spectrometry-Electron Activated Dissociation Tandem Mass Spectrometry. Anal Chem 88:3440-3
Wang, Yun Hwa Walter; Meyer, Rosana D; Bondzie, Philip A et al. (2016) IGPR-1 Is Required for Endothelial Cell-Cell Adhesion and Barrier Function. J Mol Biol 428:5019-5033
Srinivasan, Srimathi; Chitalia, Vipul; Meyer, Rosana D et al. (2015) Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis. Angiogenesis 18:449-62
Yu, Xiang; Sargaeva, Nadezda P; Thompson, Christopher J et al. (2015) In-Source Decay Characterization of Isoaspartate and ?-Peptides. Int J Mass Spectrom 390:101-109
Steinhorn, Benjamin S; Loscalzo, Joseph; Michel, Thomas (2015) Nitroglycerin and Nitric Oxide--A Rondo of Themes in Cardiovascular Therapeutics. N Engl J Med 373:277-80
Walsh, Erica M; Niu, MengMeng; Bergholz, Johann et al. (2015) Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation. Biochem Biophys Res Commun 461:293-9

Showing the most recent 10 out of 252 publications