Molecular and biological characterization of a zonula occludens-1 homologue in Hydra vulgaris, named HZO-1

Zonula occludens-1 (20-1) is one of the earliest identified molecular compo nents of tight junctions. Sequence analysis has placed ZO-1 into the broade r membrane-associated guanylate kinase (MAGUK) protein family that contains such diverse members as postsynaptic density 95 (PSD-95), Drosophila discs large tumor suppressor gene product (dlg-A), p55, and TamA. Studies in bot h vertebrates and invertebrates have established that the MAGUK family is i nvolved in a wide variety of cellular functions. These functions involve th e regulation of such cellular processes as: (1) tight junction formation, ( 2) cell proliferation, (3) cell differentiation, and (4) neuronal synapse t ransmission. Extending these studies, we report the presence of a ZO-1 homo logue in Hydra vulgaris, a member of the Cnidaria, the second oldest phylum of the animal kingdom. Hydra ZO-1 (HZO-1) is encoded by a single messenger RNA (mRNA) of approximately 6.0 kb that contains an open reading frame of 5,085 bp. The 191 kDa predicted protein consists of a characteristic MAGUK domain structure, including three PSD-95/SAP90, discs-large, ZO-1 (PDZ) dom ains, a src homology (SH3) domain, and a guanylate kinase (GUK) domain. Wes tern blot analysis using an antibody generated from a synthetic peptide des igned from the HZO-1 sequence confirmed the presence of a Hydra protein of the appropriate mass. While whole mount in situ hybridization determined th at HZO-1 mRNA was expressed along the entire longitudinal axis of Hydra, cr oss-sectional analysis established that HZO-1 mRNA expression was restricte d to the ectoderm or outer cell layer of the organism's epithelial bilayer. Consistent with this mRNA expression pattern, immunofluorescence studies l ocalized HZO-1 protein to the epical plasma membrane of ectodermal cells. I t is unclear what role HZO-1 has in the cellular physiology of Hydra; howev er, immunolocalization studies indicate a conserved plasma membrane-associa ted function(s), as reported for its counterparts in other invertebrate and vertebrate species. These studies establish that the MAGUK family of prote ins with a membrane-associated function arose early during metazoan evoluti on, even before the divergence of protostomes and deuterostomes.