Olfactomedin 2: Expression in the Eye and Interaction with Other Olfactomedin Domain-Containing Proteins

Olfactomedin was described almost 20 years ago as a novel 57-kDa glycoprotein exclusively expressed in the frog olfactory neuroepithelium.1 Subsequent experiments by many laboratories demonstrated that olfactomedin contains a domain in its C terminus that is present in many proteins in species ranging from reef-building coral Acropora millepora (the phylum Cnidaria) to Homo sapiens.2–7 This domain has a length of approximately 250 amino acids and was named the olfactomedin domain. There are at least 13 proteins containing the olfactomedin domain in mammals, and these proteins form a family.4,8,9 This family segregates into seven subfamilies on the basis of domain organization, biochemical properties, and expression patterns.4 The most studied olfactomedin domain–containing protein is myocilin. Mutations in the Myocilin gene are found in more than 10% of juvenile open-angle glaucoma cases and in 35% to 4% of patients with adult-onset primary open-angle glaucoma.10–14 Myocilin proteins form subfamily III on a phylogenetic tree.4,9 Three proteins, olfactomedin 1 (Olfm1), olfactomedin 2 (Olfm2), and olfactomedin 3 (Olfm3), form subfamily I. Olfm1 is also known as noelin in chicken and Xenopus,15,16 pancortin in mice,17 olfactomedin-related glycoprotein in rats,18 and hOlfA in humans.5 Olfm2 is also known as OlfC,5 and Olfm3 is also known as optimedin.19 These proteins are the most conserved among other olfactomedin domain–containing proteins. High conservation of the amino acid sequences implies that Olfm1, -2, and -3 proteins have important functions. Indeed, overexpression of Olfm1 during embryonic development causes an excess of neural crest emigration in chicken,15 leads to an expansion of the neural plate and enlargement of the neural tube and retina in Xenopus,20 increases the thickness of the optic nerve and produces a more extended projection field in the optic tectum in zebrafish.21 Inhibition of olfm1 expression by olfm1-specific morpholino oligonucleotides reduces eye size, inhibits optic nerve extension, and increases the number of apoptotic cells in the retinal ganglion cell and inner nuclear layers.21 At the same time, elimination of the central (M region) part of Olfm1 in mice produces a relatively mild phenotype.22 It has been shown that knockdown of olfm2 protein expression by morpholino oligonucleotides produces a phenotype that is very similar to that described for the olfm1 knockdown in zebrafish.23 Genetic data indicate that mutation of the OLFM2 gene in humans leading to the R144Q substitution in the protein sequence is a possible disease-causing mutation in Japanese patients with open-angle glaucoma.24 The T86M substitution in the OLFM2 protein has been associated with colorectal cancers.25 Overexpression of Olfm3 inhibits neurite outgrowth and induces Ca2+-dependent aggregation of NGF-stimulated PC12 cells.26 It has been suggested that the expression of Olfm3 stimulates the formation of adherens and tight junctions and modulates cytoskeletal organization, cell–cell adhesion, and cell migration in the brain and retina.26 In this study, we investigated the properties of mammalian OLFM2. Olfm2 and -1 showed an overlapping expression pattern in the course of eye development in the rat. OLFM2 interacted with Olfm1 and -3 but not with the more distantly related proteins myocilin and gliomedin. Although the R144Q and T86M substitutions did not inhibit secretion of OLFM2, L420S substitution in the olfactomedin domain of OLFM2, which corresponds to the I477S mutation in human myocilin, inhibited its secretion. Moreover, secretion of wild-type Olfm1 or -3, but not myocilin, was inhibited in the presence of this mutation. We suggest that severe mutations in one of the closely related olfactomedin domain–containing proteins (Olfm1-3) blocks secretion and probably activity of the whole subfamily.