Identification and characterization of AMACO, a new member of the von Willebrand factor A-like domain protein superfamily with a regulated expression in the kidney.
The genes coding for human and mouse AMACO, an extracellular matrix protein containing VWA-like domains related to those in MAtrilins and COllagens, were detected in databases, the cDNAs were cloned, and the primary structures were deduced from the nucleotide sequences. The genes consist of 14 exons and have a similar exon/intron organization. The protein consists of a signal peptide sequence, an N-terminal VWA domain connected to two additional, tandem VWA domains by a cysteine-rich sequence and an epidermal growth factor (EGF)-like domain. The C terminus is made up of another EGF-like domain followed by a unique sequence present in mouse, but absent in human. The predicted molecular weight of the proteins is 79,485 in human and 83,024 in mouse. Full-length AMACO was expressed in 293-EBNA cells, purified by use of an affinity tag and subjected to biochemical characterization. Both monomers and aggregates of AMACO were recovered, as shown by electron microscopy and SDS-PAGE. AMACO was found in the media of a variety of established cell lines of both fibroblast and epithelial origin. In the matrix formed by 293-EBNA cells overexpressing the protein, AMACO was deposited in patchy structures that were often cell-associated. Affinity-purified antibodies detect expression in cartilage and expression associated with certain basement membranes. In the kidney of adult mice, a second promoter located in intron 4 is active. If the resulting transcript is translated it could not yield a secreted protein because of the lack of a signal peptide sequence. The developmental switch from an AMACO mRNA, expressed by the newborn kidney, to the truncated transcript found in the adult kidney indicates an unusual regulation of AMACO expression.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism; D014408:Biomarkers, Tumor; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins; D002356:Cartilage / Q000378:metabolism; D002460:Cell Line; D019902:Chondrocytes / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry; D004847:Epithelial Cells; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D017403:In Situ Hybridization; D015603:Keratinocytes / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008168:Lung / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014157:Transcription Factors; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D016376:Oligonucleotides, Antisense; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; C496188:VWA2 protein, human; C480232:Vwa2 protein, mouse; D014841:von Willebrand Factor; D004815:Epidermal Growth Factor; D003545:Cysteine