Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder causedby mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence ofunsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visualimpairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressedand exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of theKSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in culturedprimary keratinocytes (n¼3) and keratocytes (n¼4). Immunohistochemistry, semiquantitative RT-PCR, insitu RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneasKS was predominantly found in the stroma, but absent, or barely detectable, in the corneal epithelium.A similar pattern of distribution was found in the epidermis, with KS mainly localised in the derma. Asexpected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues ontoKSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and inthe endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytesexpress and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarlyexpression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromalkeratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and expressvery little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare geneticdisorder must therefore involve and target stromal keratocytes. If coupled to the accuracy of HPLC-based assay that we developed to determine the amount of KS in serum, our findings could lead to more targetedtherapeutic treatments of the ocular features in MCD patients.
Localization and expression of CHST6 and keratan sulfate proteoglycans in the human cornea / Di Iorio, E; Barbaro, V; Volpi, Nicola; Bertolin, M; Ferrari, B; Fasolo, A; Arnaldi, R; Brusini, P; Prosdocimo, G; Ponzin, D; Ferrari, S.. - In: EXPERIMENTAL EYE RESEARCH. - ISSN 0014-4835. - STAMPA. - 91:2(2010), pp. 293-299. [10.1016/j.exer.2010.06.001]
Localization and expression of CHST6 and keratan sulfate proteoglycans in the human cornea
VOLPI, Nicola;
2010
Abstract
Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder causedby mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence ofunsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visualimpairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressedand exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of theKSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in culturedprimary keratinocytes (n¼3) and keratocytes (n¼4). Immunohistochemistry, semiquantitative RT-PCR, insitu RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneasKS was predominantly found in the stroma, but absent, or barely detectable, in the corneal epithelium.A similar pattern of distribution was found in the epidermis, with KS mainly localised in the derma. Asexpected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues ontoKSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and inthe endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytesexpress and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarlyexpression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromalkeratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and expressvery little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare geneticdisorder must therefore involve and target stromal keratocytes. If coupled to the accuracy of HPLC-based assay that we developed to determine the amount of KS in serum, our findings could lead to more targetedtherapeutic treatments of the ocular features in MCD patients.
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