A model of FAS 1 domain 4 of the corneal protein β igh 3 gives a clearer view on corneal dystrophies

βig-h3 (TGFBIP, kerato-epithelin, MP78/70, RGD-CAP) was first identified as a TGF-β inducible gene in a lung adenocarcinoma cell line [1]. The cDNA sequence encodes a secreted protein of 68 kDa molecular mass, consisting of four regions of internal homology of approximately 140 amino acids and a carboxy terminal Arg-Gly-Asp (RGD) motif, which in other proteins mediates binding to receptors of the integrin family [2]. The tandemly repeated regions are known as FAS1 domains due to the original discovery of four such domains in the insect cell adhesion molecule fasciclin I [3] (Figure 1). Interest in βig-h3 has increased sharply since genetic studies discovered that a group of hereditary corneal dystrophies are caused by specific missense mutations in the BIGH3 (TGFBI) gene [4]. Despite the genetic characterization of these, BIGH3 related corneal dystrophies, the biochemical mechanisms responsible for the alteration of protein behavior remain unclear. The likely effects of amino acid substitutions can be predicted only if a three-dimensional structure of the affected protein is available. The recently determined structure of a fasciclin I fragment, consisting of two FAS1 domains, provides a template for the entire FAS1 domain superfamily [5]. The βig-h3 protein closely resembles fasciclin I in that it consists of an uninterrupted tandem of 4 FAS1 domains. Here this new structural information was used to build a homology model of the βig-h3-FAS1 domain 4 (the disease-causing mutations are clustered in this domain), and a detailed examination was carried out. Before describing the results of this analysis an introduction is given to the βig-h3 protein and the corneal dystrophies resulting from BIGH3 mutations. Expression and biochemical properties of βig-h3: The BIGH3 gene is expressed by a large number of human tissue types, including the corneal epithelium of the eye [1,6]. Despite the initial characterization of βig-h3 as a TGF-β-induced gene product [1], the expression of βig-h3 is not induced by TGF-β in corneal epithelial cells, where there is an undetectable level of TGF-β mRNA [6]. A study of the embryonic expression of the mouse BIGH3 gene has shown that it is expressed in the mesenchyme of most developing organs and the developing tissues of the heart, pancreas and eye [7]. The expression pattern of βig-h3 in developing bovine tissues resembles that of type VI collagen and, although less widely distributed than type VI collagen, the βig-h3 protein is prominently present in the extracellular matrix of a wide range of developing and mature tissues [8]. For example, βig-h3 is found in skin, especially the papillary dermis [9,10]. In normal corneas (Figure 2), the βig-h3 protein is detected throughout the stroma, in Bowman’s layer and in endothelial cells [6,8,11-13]. In healing and diseased corneas, βig-h3 has been identified in the retrocorneal fibrous membrane (which develops posterior to the pre-existing Descemet’s membrane) in the stromal keratocytes [13,14], and as deposits in the corneal stroma in the case of corneal dystrophies [12]. It appears that βig-h3 has a role in corneal development and healing, as well as additional functions in other tissues [15-17], but the precise physiological role of βig-h3 is still largely unknown. Biochemical studies suggest that βig-h3 may © 2003 Molecular Vision