Enzymatic Activity of Mutant Sulfotransferases Found in Macular Corneal Dystrophy Type II

Abstract

Purpose:: In human, CHST6 encodes corneal GlcNAc 6-O sulfotransferase (hCGn6ST, also known as GlcNAc6ST-5 and GST4b), which is an essential enzyme for production of sulfated keratan sulfate (KS), and mutation on the gene leads to a hereditary eye disease, macular corneal dystrophy (MCD), which the patients develop clouding cornea. MCD is categorized into 3 subtypes by immunological examinations; no sulfated KS in cornea and serum (type I), presence of KS in cornea and serum, even at much reduced level in some case (type II), and no KS in serum and corneal matrix but present in stromal keratocytes (type IA). From our finding of DNA rearrangements at putative promoter/enhancer region of CHST6 on genomes of MCD type II patients, we hypothesized that complete lack of hCGn6ST activity leads to MCD type I phenotype and cornea-specific loss of CHST6 expression results in MCD type II phenotype. Different from our hypothesis, several missense mutations on CHST6 have been reported to be responsible for MCD type II. In this study, we examined sulfotransferase activity of reported mutant hCGn6STs and asked whether the mutants have ability to produce sulfated KS.

Methods:: Employing site-directed mutagenesis, we prepared expression vectors encoding mutant hCGn6STs that were reported to be candidate mutations for MCD type II, and evaluated their sulfotransferase activity as ability to produce 5D4-positive highly sulfated KS in HeLa cells. We also prepared soluble form of the mutant enzymes and analyzed their sulfotransferase activity over synthetic carbohydrate substrate in vitro.

Results:: Among 6 mutant enzymes analyzed, we found that 2 enzymes had weaker but significant sulfotransferase activity and produced 5D4-positive KS. From the result and reported mutation information of MCD type II, we confirmed that all the MCD type II patients had potentially active sulfotransferases on their genomes.

Conclusions:: MCD type II patients have active hCGn6ST, at least on one allele, on their genomes and presence or absence of active hCGn6ST determines biochemical differences of MCD subtypes. MCD type II may be caused by tissue-specific suppression of CHST6 expression, as we hypothesized before, or by very weak enzymatic activity of mutant hCGn6ST, which produces minimal amount of 5D4-positive highly sulfated KS but not sufficient activity for preventing production of unusual carbohydrate deposits such as low-sulfated KS.

Keywords: enzymes/enzyme inhibitors • mutations • proteoglycans/glycosaminoglycans