6-0-sulfated polysaccharides and methods of preparation thereof

Abstract

Disclosed are methods of 6-O sulfating glucosaminyl N-acetylglucosamine residues (GlcNAc) in a polysaccharide preparation and methods of converting anticoagulant-inactive heparan sulfate to anticoagulant-active heparan sulfate and substantially pure polysaccharide preparations made by such methods. Also disclosed is a mutant CHO cell which hyper-produces anticoagulant-active heparan sulfate. Methods for elucidating the sequence of activity of enzymes in a biosynthetic pathway are provided.

1 . A mutant CHO cell that produces more than 28% HS act , relative to HS total . 
     
     
         2 . A mutant CHO cell that produces between 28% and 50% HS act , relative to HS total . 
     
     
         3 . The mutant CHO cell of  claim 1 , produced by a method comprising:
 (a) transforming a CHO cell with multiple copies of 3-OST-1;   (b) mutagenizing the cell obtained in step (a);   (c) isolating a mutant cell from step (b) which fails to produce HS act ; and   (d) transforming the cell obtained in step (c) with 6-OST.   
     
     
         4 . A method of identifying components in a biosynthetic pathway comprising the steps of:
 a) providing a target cell which expresses at least the upstream components of the biosynthetic pathway;   b) transforming the target cell with multiple copies of an isolated biosynthetic pathway downstream gene;   c) mutagenizing the transformed target cell; and   d) identifying transformed and mutagenized target cells that fail to express the phenotype characteristic of the biosynthetic pathway.   
     
     
         5 . The method of  claim 4 , further comprising the step
 (e) correcting the step (d) cells, wherein the corrected cells express the wild-type phenotype of the cell in step (a).   
     
     
         6 . The method of  claim 5 , wherein the correcting step comprises transforming the cell with the nucleic acid that encodes an upstream gene. 
     
     
         7 . The method of  claim 6 , wherein the upstream gene is a cDNA or genomic DNA. 
     
     
         8 . The method of  claim 4 , wherein the cells of step (d) are transformed with a pool of preselected cDNAs for components of the biosynthetic pathway. 
     
     
         9 . The method of  claim 4 , wherein the cells of step (d) are transformed with a cDNA library derived from a cell that expresses wild-type phenotype. 
     
     
         10 . The method of  claim 5 , wherein the correcting step comprises contacting the cells of step (d) with the gene product of an upstream gene. 
     
     
         11 . The method of  claim 4 , further comprising the step of isolating the cells from step (d). 
     
     
         12 . The method of  claim 4 , further comprising the step of analyzing the cells of step (d). 
     
     
         13 . The method of  claim 7 , further comprising the step of isolating the upstream gene in the biosynthetic pathway. 
     
     
         14 . The method of  claim 4 , wherein the mutagenesis step comprises a mutagenesis technique selected from the group consisting of chemical mutagenesis, ion radiation, and ultraviolet radiation. 
     
     
         15 . The method of  claim 4 , wherein the step of identifying the gene cDNA comprises complementation analysis. 
     
     
         16 . The method of  claim 4 , wherein the identifying step comprises identifying the gene by a teclmique selected from the group consisting of Northern blot analysis, Southern blot analysis, and Western blot analysis. 
     
     
         17 . The method of  claim 4 , wherein the identifying further comprises isolating of the gene using PCR.