Heterogeneity of Heparan Sulfates in Human Lung

Abstract

Heparan sulfates (HS), a class of glycosaminoglycans, are long linear complex polysaccharides covalently attached to a protein core. The HS molecules are made up of repeating disaccharides onto which modification patterns are superimposed. This results in a large structural heterogeneity and forms the basis of specific interactions of HS toward a vast array of proteins, including growth factors and proteases. To study HS heterogeneity in the lung, we used phage display technology to select seven antibodies against human lung HS. Antibodies reacted with HS/heparin, but not with other glycosaminoglycans or polyanions. Sulfate groups were essential for antibody binding. The amino acid sequence of the antibodies was established, the complementarity determining region 3 of the heavy chain containing basic amino acids. The antibodies defined HS epitopes with a characteristic tissue distribution. Antibody EV3A1 primarily stained macrophages. Other antibodies primarily stained basement membranes, but with different preference toward type of basement membrane. Antibody EV3C3 was the only antibody which clearly reacted with bronchiolar epithelial cells. In human lung parenchyma, basic fibroblast growth factor and vascular endothelial growth factor were largely bound by HS. Some antibodies blocked a basic fibroblast growth factorbinding site of HS, and one antibody blocked a vascular endothelial growth factor-binding site of heparin. Taken together, these data suggest a specific role for HS epitopes in human lung. The antibodies obtained may be valuable tools to study HS in pulmonary diseases.Heparan sulfates (HS) are members of the glycosaminoglycan (GAG) family, consisting of repeating disaccharide units onto which modification patterns are superimposed. HS bind and modulate a myriad of molecules, including growth factors, cytokines, proteases, antiproteases, matrix molecules, and viral and bacterial proteins (1,2). This large number of interactions suggests an extensive structural variation within HS. The structural diversity of HS is brought about by specific chain modifications during the biosynthesis of HS, including deacetylation, sulfation, and epimerization. The addition of, e.g., sulfate groups leads to the generation of specific motifs that make HS highly versatile, protein-

Role of the Heparan Sulfate Proteoglycan Syndecan-1 (CD138) in Delayed-Type Hypersensitivity

“…Cryosections (3 m) of ear tissue from Sdc-1 KO and WT mice were dried and blocked for 10 min with PBS containing 2% BSA. The sections were subsequently incubated with the primary single-chain Fv Abs HS4C3 (28) or EV3C3 (29), the rat-anti-mouse Sdc-1 mAb 281-2, or its isotype control Ab (BD Biosciences) for 90 min at 22°C. Bound Abs were visualized using anti-VSV-tag Ab P5D4 followed by Alexa Fluor 488-labeled anti-mouse or rat IgG Abs (Invitrogen).…”

“…Sdc-1 modulates the activity of a variety of proteins relevant to inflammation via interactions with its heparan sulfate chains (7)(8)(9), which are capable of forming specific ligand binding sites depending on posttranslational modifications such as epimerization and N-and O-sulfation (10,13). To uncover potential structural changes in heparan sulfate during the course of the DTH reaction, we used the heparan sulfate-specific single-chain Ab EV3C3 (29) and the Ab HS4C3 (28), which preferentially recognizes a 3-O-sulfated heparan sulfate motif and which is able to compete with the anti-thrombin III binding site of heparin (33), for the immunolocalization of specific heparan sulfate epitopes in cryosections of vehicle-and oxazolone-treated ears of Sdc-1 KO and WT mice (Fig. 4).…”

Plasminogen-Mediated Activation and Release of Hepatocyte Growth Factor from Extracellular Matrix

“…Internal proteolytic cleavage is required to generate the active disulfide-linked dimer. Furthermore, secreted HGF becomes tightly bound to the heparan sulfate moiety of proteoglycans (25) that are abundant in extracellular matrices within the lung (26). Bound HGF must be released from these sites of sequestration so that it can access the c-met receptor on neighboring target cells (27).…”

Irreversible Glucuronyl C5-epimerization in the Biosynthesis of Heparan Sulfate

“…The HS generated in a single tissue (human aortic wall) shows gradual structural change with increasing age of the individual (21,22). Immunohistochemical application of various anti-HS antibodies demonstrate cellspecific expression of HS epitopes of different structure (23,24). Regulation presumably applies also to the GlcA-IdoA conversion in HS biosynthesis, judging from the variable content and domain distribution of IdoA units in HS chains.…”