Localization and expression of CHST6 and keratan sulfate proteoglycans in the human cornea.
Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder caused by mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence of unsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visual impairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressed and exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of the KSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in cultured primary keratinocytes (n = 3) and keratocytes (n = 4). Immunohistochemistry, semiquantitative RT-PCR, in situ RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneas KS 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. As expected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues onto KSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and in the endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytes express and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarly expression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromal keratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and express very little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare genetic disorder 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 targeted therapeutic treatments of the ocular features in MCD patients.
The Veneto Eye Bank Foundation, Via Paccagnella 11, c/o Padiglione Giovanni Rama, 30174 Zelarino-Venice, Italy.
Enzo
E
Di Iorio
Vanessa
V
Barbaro
Nicola
N
Volpi
Marina
M
Bertolin
Barbara
B
Ferrari
Adriano
A
Fasolo
Renato
R
Arnaldi
Paolo
P
Brusini
Giovanni
G
Prosdocimo
Diego
D
Ponzin
Stefano
S
Ferrari
D000818:Animals; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003315:Cornea / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007632:Keratan Sulfate / Q000235:genetics / Q000378:metabolism; D000071234:Lumican; D051379:Mice; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism*; D041701:Swiss 3T3 Cells; D000097811:Carbohydrate Sulfotransferases
D011508:Chondroitin Sulfate Proteoglycans; D036341:Intercellular Signaling Peptides and Proteins; D007632:Keratan Sulfate; D000071234:Lumican; D011509:Proteoglycans; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C490948:KERA protein, human; C000604306:LUM protein, human; C000604307:Lum protein, mouse; C063488:OGN protein, human