Apr 20th, 1:00 PM - 4:00 PM
Analysis of Sulfated Glycosaminoglycan Binding Sites Within Type XI Collagen
Sulfated glycosaminoglycans such as chondroitin sulfate are unbranched polysaccharides. Chondroitin sulfate contains repeating disaccharide subunits composed of D-glucuronate and N-acetyl-Dglalactosamine sulfate, whereas heparin sulfate consists of repeating disaccharide subunits of L-iduronate-2-sulfate and N-sulfo-D-glucosamine- 6-sulfate. Chondroitin sulfate and heparin sulfate are located primarily in the extracellular matrix of cells. The functions of chondroitin sulfate and heparin sulfate are not well understood. Chondroitin sulfate and heparin sulfate are found to interact with proteins, and may play structural roles within the body. Type XI Collagen is a minor constituent of the extracellular matrix of cartilage and is essential in the regulation of collagen fibril assembly and diameter. The alpha 1 chain of Collagen XI (Collagen α1(XI) ) contains a variable region that is modulated by alternative splicing in a tissue-specific and developmental manner. Preliminary data suggests that some Collagen XI isoforms bind glycosaminoglycans. Biochemical and biophysical methods will be used to assess the interactions between Collagen α1(XI) and the glycosaminoglycans heparin sulfate and chondroitin sulfate. Collagen α1(XI) isoforms will be expressed in Escherichia coli and then purified using affinity chromatography. Circular dichroism spectropolarimetry will be used to monitor protein secondary structure. Solid phase binding data and surface plasmon resonance spectroscopy will be used to evaluate the kinetics of interactions between the Collagen α1(XI) isoforms and the glycosaminoglycans. Analytical ultracentrifugation and multi angle laser light scattering will be used to assess the hydrodynamic and physical properties of the protein glycosaminoglycan complex. Information gained from this study will help us to understand how Collagen α1(XI) isoforms interact with glycosaminoglycans and contribute to cell-matrix interactions as well as matrix-matrix interactions important in development and in the progression of disease.