Matrix Biochemistry
contact: A. van Kuppelvelt A.vanKuppevelt@ncmls.ru.nl
Background
Glycosaminoglycans (GAGs) are long, unbranched polysaccharides, most of which are covalently linked to a protein core to form proteoglycans. Depending on the nature of their backbone, one can discern the class of galactosaminoglycans: chondroitin sulfate (CS) and dermatan sulfate (DS), and glucosaminoglycans: heparan sulfate (HS) and heparin. Superimposed upon the backbone is a pattern of sulfation and acetylation modifications that constitute different domains. The resulting unique modification �sequences� on GAG molecules are instrumental in the binding of various biologically active proteins. Through the highly regulated affinity towards effector molecules, GAGs are important modulators of numerous biological processes. However, the sequence-specific recognition of a GAG sequence has only been shown for the serine protease inhibitor Antithrombin-III, whereas less-defined preferential binding requirements have been postulated for some growth factors, e.g. FGF-1 and FGF-2. Investigating the exact role(s) of GAGs in physiological and pathological processes has historically been hampered by a lack of appropriate tools.
Research
Using phage display, we have generated a large panel (>100) of antibodies against CS, DS, HS, and heparin epitopes. These antibodies were selected from a semi-synthetic human �single chain variable fragment� library (scFv library #1). ScFv antibodies are comprised of the variable (antigen binding) domains of both the light and the heavy antibody chain. Selections were performed against a variety of GAGs from commercial and biological sources.
Main areas of research-interest within our lab are:
1. The roles of GAGs in renal (patho)physiology
2. The involvement of GAGs in lung and diaphragm of patients with chronic obstructive pulmonary disease (COPD)
3. The occurrence and function(s) of GAGs in ovary tumorogenesis
4. The involvement of proteoglycans, notably their GAG moieties, in skeletal muscle physiology and pathologies
Anti-GAG antibodies were used to probe changes in the topological distribution of GAG epitopes in various biological systems. Studying the topological distribution of the epitopes that are recognized by our antibodies thus enables us to get insight in potential biological roles of these GAG epitopes.
The initial characterization of the GAG epitopes recognized by our antibodies, using ELISA, inhibition ELISA, immuno precipitation, and SDS Page based techniques, has identified some of the general characteristics of the GAG sequences involved. In close collaboration with leaders in the field of GAG preparation and analysis, we are currently implementing recently acquired expertise and tools for the use of high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and mass spectrometry (MS) to determine the exact chemical composition of these GAG epitopes.
The biological functions of GAGs can also be analyzed using our anti-GAG antibodies: Heparin-specific antibodies display different affinities towards the Antithrombin-III pentasaccharide sequence and interfere to different extents with heparin anticoagulant activity. The endogenous expression of anti-HS antibodies by myogenic cells results in a functional knock-out of specific HS epitopes, which severely impairs ion housekeeping.
Perspective
Our panel of anti-GAG antibodies provides a set of unique and highly versatile tools to study GAG structure and function. As illustrated here, these antibodies can be used for immuno-histological analysis of GAG distribution, probing of biological activities, and for the purification and structural characterization of specific GAG epitopes in a variety of physiological and pathological systems.
