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Q. Chen, K.G. Shadrach, J.G. Hollyfield; SPACRCAN Binding to Hyaluronan: Molecular and Biochemical Studies . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3514.
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Purpose:: Hyaluronan (HA) is a glycosaminoglycan (GAG) in the interphotoreceptor matrix (IPM). It has been implicated as providing a primary scaffold in the IPM. Some proteins in IPM, such as SPACR and SPACRCAN, have been shown to possess HA binding motifs. Both of them have been demonstrated to bind to HA. However, there is no direct evidence that the binding is through interaction with the HA binding motif. The purpose here is to initiate a study to demonstrate the function of HA motifs in the HA binding identified in mouse SPACRCAN. Methods: : A short polypeptide fragment of mouse SPACRCAN containing each HA binding motif was subcloned into the pGEX-2TK vector and expressed in E. coli BL21 cells. Proteins purified from the cell extracts were subjected to CPC precipitation analysis in which binding of a cationic detergent, cetylpyridinium chloride, to anionic GAGs like HA leads to co-precipitation of proteins interacting with the GAGs. To demonstrate the binding was HA specific, digestion with a HA-specific hyaluronidase, Streptomyces hyaluronidase, was included in the co-precipitation analysis. Results: Polypeptides containing four of the HA binding motifs in mouse SPACRCAN have been expressed in E. coli BL21 cells. CPC precipitation analysis showed that the polypeptides were precipitated in the pellets. However, pre-incubation of the polypeptides with Streptomyces hyaluronidase dramatically decreased the amount of peptides precipitated in the pellets. Conclusions: The HA binding motif containing polypeptides expressed in E. coli binds to HA. Further mutagenesis studies are in progress to determine the HA binding is through the HA binding motifs.
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