Identification and preliminary characterization of mouse Adam33

Abstract

Background: The metalloprotease-disintegrin family, or ADAM, proteins, are implicated in cell-cell interactions, cell fusion, and cell signaling, and are widely distributed among metazoan phyla. Orthologous relationships have been defined for a few ADAM proteins including ADAM10 (Kuzbanian), and ADAM17 (TACE), but evolutionary relationships are not clear for the majority of family members. Human ADAM33 refers to a testis cDNA clone that does not contain a complete open reading frame, but portions of the predicted protein are similar to Xenopus laevis ADAM13.

Results: In a 48 kb region of mouse DNA adjacent to the Attractin gene on mouse chromosome 2, we identified sequences very similar to human ADAM33. A full-length mouse cDNA was identified by a combination of gene prediction programs and RT-PCR, and the probable full-length human cDNA was identified by comparison to human genomic sequence in the homologous region on chromosome 20p13. Mouse ADAM33 is 44% identical to Xenopus laevis ADAM13, however a phylogenetic alignment and consideration of functional domains suggests that the two genes are not orthologous. Mouse Adam33 is widely expressed, most highly in the adult brain, heart, kidney, lung and testis.

Conclusions: While mouse ADAM33 is similar to Xenopus ADAM13 in sequence, further examination of its embryonic expression pattern, catalytic activity and protein interactions will be required to assess the functional relationship between these two proteins. Adam33 is expressed in the mouse adult brain and could play a role in complex processes that require cell-cell communication.

Figure 1

Adam33 structure and phylogeny (A) Location and orientation of genes on BAC389B9. The entire Adam33 gene is contained within a 48 kb contig (AF155960) that also contains the Gfra4 gene and the 3' ends of Atrn and Sn. (B) Beginning with the translational start site, Adam33 is contained within 22 exons that span approximately 12.6 kb of genomic sequence. The putative signal sequence cleavage site (arrow), metalloprotease domain (asterisks) and transmembrane domain (line) are indicated. (C) ClustalW Phylogeny for ADAM proteins most closely related to Adam33 (arrow). For reference, human (Homo), mouse (Mus), and Xenopus laevis (Xen.) orthologs are shown for Adam9 and Adam22.

Figure 2

Amino acid similarity among mouse (m) and human (h) ADAM33 proteins, and Xenopus laevis (X) ADAM13. Residues identical to the mouse protein are indicated in bold. Putative signal sequence cleavage sites (SS) are indicated by arrow. The metalloprotease site (MP), disintegrin loop (DL) and transmembrane domain (TM) are boxed and cysteine residues are highlighted by black boxes. The metalloprotease (MPD), disintegrin-like (DD), cysteine-rich (CRD), and EGF-like (EGF) domains are indicated.

Figure 3

Mouse Adam33 expression. (A) Northern hybridization assay of mouse Adam33 (using IMAGE clone 636599) on polyadenylated RNA from adult mouse tissues. (B) Expression profile of mouse Adam 33 in Origene Mouse Rapid-Scan cDNA panel. Panel consists of normalized cDNA from 24 mouse tissues or embryo stages, serially diluted over a 4-log range (1000X-1X), where the lowest concentration (IX) is approximately 1 pg. Adam33 expression was assayed by PCR amplification of a 368 bp fragment of the mouse Adam33 cDNA sequence (band shown). Lane 1: brain; 2: adrenal gland; 3: heart; 4: pancreas; 5: kidney; 6: uterus; 7: spleen; 8: prostate gland; 9: thymus; 10: day 8.5 embryo; 11: liver; 12: day 9.5 embryo; 13: stomach; 14: day 12.5 embryo; 15: small intestine; 16: day 19 embryo; 17: muscle; 18: virgin breast; 19: lung; 20: pregnant breast; 21: testis; 22: lactating breast; 23: skin; 24: involuting breast. Slight variation in the apparent mobility among lanes is due to sample differences; fragments of identical mobility among different tissue samples were produced when individual RT-PCR reactions were repeated. Absence of signal in the 1000× but presence in the 100× dilutions for two of the tissues are likely to be caused by contaminants in the RNA preparation that inhibited the RT-PCR reaction.

Figure 4

Expression pattern of Adam33 in adult mouse brain. (A, D) Photomicrographs of coronal sections of adult mouse brain, from the Mouse Brain Library [25],[26]. Boxes indicate approximate regions shown in panels B-C and E-F. (B-C and E-F) In situ hybridization of digoxigenin labeled antisense (B, E) and sense (C, F) RNA probes derived from IMAGE clone 636599. Adam33 expression was observed only in the dentate gyrus (DG) and pyramidal cell layer (Py) of the hippocampus (panel B) and the granule layer (Gr) of the cerebellum (panel E), indicated by arrows.