Isolation and Characterization of Heparan Sulfate from Human Lung Tissues

Abstract

Glycosaminoglycans are a class of linear, highly negatively charged, O-linked polysaccharides that are involved in many (patho)physiological processes. In vitro experimental investigations of such processes typically involve porcine-derived heparan sulfate (HS). Structural information about human, particularly organ-specific heparan sulfate, and how it compares with HS from other organisms, is very limited. In this study, heparan sulfate was isolated from human lung tissues derived from five donors and was characterized for their overall size distribution and disaccharide composition. The expression profiles of proteoglycans and HS-modifying enzymes was quantified in order to identify the major core proteins for HS. In addition, the binding affinities of human HS to two chemokines-CXCL8 and CCL2-were investigated, which represent important inflammatory mediators in lung pathologies. Our data revealed that syndecans are the predominant proteoglycan class in human lungs and that the disaccharide composition varies among individuals according to sex, age, and health stage (one of the donor lungs was accidentally discovered to contain a solid tumor). The compositional difference of the five human lung HS preparations affected chemokine binding affinities to various degrees, indicating selective immune cell responses depending on the relative chemokine-glycan affinities. This represents important new insights that could be translated into novel therapeutic concepts for individually treating lung immunological disorders via HS targets.

Keywords: chemokine/GAG interactions; chemokines; disaccharide composition; glycosaminoglycans; heparan sulfate; lung.

Figure 1

Expression profile of various genes in human lung tissues determined by RT-qPCR. Relative patient-specific mRNA expression levels of relevant syndecans (SDC) 1,2,4; glypicans (GPC) 3,4,5; sulfatases (SULF) 1,2; O-sulfotransferases (OST) 2OST, 3OST-1, 3 OST-3b; and N-sulfotransferase (NDST) 1,2. mRNA of five different donors are depicted in relation to the housekeeping gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) as mean ± SD with n = 3.

Figure 2

Size exclusion chromatogram of the different HS isolates from donor lungs (f80t, f80, m70, f92, m53, f89). Samples starting with f are from female patients, and those with m are from male, with the number depicting the age of the lung and the t identifying tumor lobe. Chromatograms are compared with two commercially available porcine-derived HS (cHS1 and cHS2). Data were recorded using a diode array detector, but the absorption maximum at 211 nm was used for the HS preparations and standards because of the highest sensitivity at this wavelength. The AUC of the HS preparations of the m53 and f89 differ significantly from the preparations of the other samples because different amounts of process-related impurities are expected to contribute to the absorption of the samples at 211 nm.

Figure 3

(A–G) Relative molar abundance (in percentage) of disaccharide composition of the different HS preparation from donor’s lungs, derived by SAX-HPLC, depicted as mean ± SEM. Data sets were compared using Student’s t-test. * p < 0.05, ** p < 0.01 was considered as statistically significant. (f refers to females, m to males, t to tumor lobe, and the number represents the donor’s age). Mean (± SEM) was calculated from three independent SAX-HPLC runs. The obtained AUCs were measured for each peak and compared with AUCs of commercially available HS disaccharide standards (Iduron).

Figure 4

Isothermal fluorescence titration of CXCL8 and CCL2 with commercially available HS derived from porcine compared with HS prepared from lung tissue of different humans. With f for females, m for males, t for tumor lobe, and the number for the patient’s age. Data are shown as mean ± SEM of three measurements; groups were compared with the corresponding titration using Student´s t-test with * cHS1 or ^o between normal versus tumor lung lobe; */^o p < 0.05, ** p < 0.01.