Isolation and Identification of Glycosaminoglycans

Abstract

The isolation and identification of glycosaminoglycans capable of binding to proteins having a heparin-binding domain is disclosed, as well as the use of the glycosaminoglycans isolated in the growth and/or development of tissue.

1 - 16 . (canceled) 
     
     
         17 . A method for preparing a heparan sulfate composition enriched for heparan sulfate molecules capable of specific and high affinity binding to a protein having a heparin-binding domain, wherein the heparin-binding domain is capable of binding to heparan sulfate, the method comprising:
 (i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide molecules each consist of the heparin-binding domain and optionally 1-20 additional amino acids at one or each of the N- and C-terminal ends;   (ii) contacting the solid support under low salt conditions with a starting mixture enriched for heparan sulfate that has been isolated from the core protein component of heparan sulfate proteoglycan, such that specifically bound heparan sulfate complexes are allowed to form between the heparin-binding domain and heparan sulfate molecules capable of specific and high affinity binding to the heparin-binding domain;   (iii) partitioning the specifically bound heparan sulfate complexes from the remainder of the mixture which does not bind the heparin-binding domain or does not specifically bind the heparin-binding domain;   (iv) dissociating the specifically bound heparan sulfate from the heparan sulfate complexes;   (v) collecting the dissociated heparan sulfate, wherein the dissociated heparan sulfate is enriched for heparan sulfate molecules that are capable of specific and high affinity binding to the heparin-binding domain, relative to the starting mixture.   
     
     
         18 . The method of  claim 17  wherein the method further comprises subjecting the collected heparan sulfate to further analysis in order to determine structural characteristics of the heparan sulfate. 
     
     
         19 . The method of  claim 17  wherein the heparan sulfate complexes are contacted with a lyase. 
     
     
         20 . The method of  claim 17  wherein the heparin binding domain is one of SEQ ID NO.s: 1-13 or 17. 
     
     
         21 . A method of identifying a heparan sulfate composition enriched for heparan sulfate molecules capable of stimulating or inhibiting the growth and/or differentiation of cells and/or tissues, the method comprising:
 (i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide molecules each consist of a heparin-binding domain, wherein the heparin-binding domain is capable of binding to heparan sulfate, and optionally 1-20 additional amino acids at one or each of the N- and C-terminal ends;   (ii) contacting the solid support under low salt conditions with a starting mixture enriched for heparan sulfate that has been isolated from the core protein component of heparan sulfate proteoglycan, such that specifically bound heparan sulfate complexes are allowed to form between the heparin-binding domain and heparan sulfate molecules that are capable of specific and high affinity binding to the heparin-binding domain;   (iii) partitioning the specifically bound heparan sulfate complexes from the remainder of the mixture which does not bind the heparin-binding domain or does not specifically bind the heparin-binding domain;   (iv) dissociating specifically bound heparan sulfate from the polypeptide-heparan sulfate complexes;   (v) collecting the dissociated heparan sulfate, wherein the dissociated heparan sulfate is enriched for heparan sulfate molecules that are capable of specific and high affinity binding to the heparin-binding domain, relative to the starting mixture;   (vi) adding the collected heparan sulfate to cells or tissues in which a protein containing the amino acid sequence of the heparin-binding domain is present;   (vii) measuring one or more of: proliferation of the cells, differentiation of the cells, expression of one or more protein markers.   
     
     
         22 . The method of  claim 21  wherein the method further comprises subjecting the collected heparan sulfate to further analysis in order to determine structural characteristics of the heparan sulfate. 
     
     
         23 . The method of  claim 21  wherein the heparan sulfate complexes are contacted with a lyase. 
     
     
         24 . The method of  claim 21  wherein the polypeptide is one of SEQ ID NO.s: 1-13 or 17.