Abstract
The complete cDNA sequence for canine ZO-2, a tight junction-specific protein, is presented. A single open reading frame encodes a polypeptide of 1,174 amino acids with a predicted molecular mass of 132,085 daltons. As noted previously (), ZO-2 is a member of the membrane-associated guanylate kinase-containing (MAGUK) protein family, a family which includes an additional tight junction-associated protein, ZO-1. These proteins contain a region homologous to guanylate kinase, an SH3 domain, and variable numbers of PSD-95/discs-large/ZO-1 (PDZ) domains, shown to be involved in protein-protein interactions. ZO-2 and ZO-1 contain three PDZ domains in the N-terminal half of the molecule. Between the first and second PDZ domains, ZO-2 displays a basic region (pI = 10.27) containing 22% arginine residues. Both ZO-1 and ZO-2 have proline-rich C-terminal regions that are not homologous to other MAGUK family members. Sequence analysis of multiple ZO-2 cDNAs reveals a 36-amino acid domain in this C-terminal region present in only some of the cDNAs. Overall, ZO-2 is highly homologous to ZO-1, showing 51% amino acid identity; however, the C-terminal ends of the molecules show only 25% amino acid identity. This suggests that the C-terminal ends of ZO-1 and ZO-2 have different functions.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) L27152
A partial sequence for ZO-2 has been characterized [1]. Analyses of these sequences indicate that ZO-1 and ZO-2 share significant homology with each other and with several other proteins, including the lethal[1]discs-large-1 tumor suppressor gene product of Drosophila [14], erythrocyte membrane-associated p55 [15], and PSD-95/SAP90, a protein found at brain presynaptic membranes [16, 17]
A comparison of the X104 aa sequence to canine ZO-2 indicates a high degree of similarity: the canine and human sequences are 87% identical at the aa level (Table II)
Summary
Library Screening—To extend the cDNA sequence for ZO-2, a hybridization probe corresponding to the 5Ј-most sequence of the partial ZO-2 cDNA [1] was generated by polymerase chain reaction using oligonucleotides 5Ј-GCA CGA GAG AGA CGG-3Ј and 5Ј-TTC ATC TTC AGG ACT-3Ј. MDCK poly(A)ϩ RNA was reverse-transcribed with the ZO-2-specific antisense linker-primer 5Ј-GAG AGA GAG AGA GAG AGA GAA CTA GTC TCG AGG TAG TCA TCG TCA TGG TC-3Ј containing an XhoI site. This primer was situated approximately 250 bp downstream of the 5Ј end of the known ZO-2 sequence contained in pM7.7. The new library was screened with an oligonucleotide containing the sequence 5Ј-TGG CTG CTG CGC CGG CTC CTC TCG CTG TAC CCG CTG CGG GCA CTT CT-3Ј end-labeled with [␥-32P]ATP (ICN Pharmaceuticals) This sequence is the complement of a known region of.
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