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

Abstract

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.The von Willebrand Factor A-like domain (VWA or vWFA) 1 is a well studied protein module present in a great variety of extracellular proteins, e.g. the prototype von Willebrand factor, the complement factors B and C2, trypsin inhibitors, collagens, matrilins, and integrins. Recently even intracellular members of the protein superfamily have been identified (for review see Ref. 1). It is generally assumed that VWA domains mediate protein-protein interactions and extracellular VWA-domaincontaining proteins are involved in cell-cell, cell-matrix, and matrix-matrix interactions and thereby play roles in important physiological processes such as immunity, hemostasis, and wound healing (2). X-ray crystallography revealed a structure made up from alternating ␣ helices and a central ␤ sheet also known as the Rossman fold. Some VWA domains contain a conserved MIDAS (metal ion-dependent adhesion site) sequence motif, which can be involved in ligand binding. The structures of the A1 domain (3) and the A3 domain (4) of the von Willebrand factor itself, and the VWA domains (I domains) of the integrin ␣ subunits ␣ L (5), ␣ M (2), ␣ 1 (6), ␣ 2 (7), and ␣ V (8), as well as that of the integrin ␤ 3 subunit (8) have been resolved. The binding sites of the vWFA1 domain for Gp1b (9) and those of integrin ␣ 2 (10) ...

Colon cancer secreted protein-2 (CCSP-2), a novel candidate serological marker of colon neoplasia

“…Human CCSP-2 mRNA and gene sequence GenBank Accession numbers as deposited by our group are, respectively, AY572972 and AY572973. The coding sequence of the colonic CCSP-2 agrees with the recently described sequence of AMACO, a transcript found as transiently expressed in the developing kidney (GenBank entry NM-198496) (Sengle et al, 2003).…”

Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

“…Protein bands were cut out and their N-terminal amino acid sequences determined in a Procise Protein Sequencer (Applied Biosystems). Peptide mass fingerprinting (27) and MALDI-TOF mass spectrometry (5, 27) were performed as described. For mass determination of the processed matrilin-3 coiled-coil domains, the affinity purified proteins were reduced in 8 M urea, 10 mM dithiothreitol at 80°C for 20 min, alkylated with 20 mM iodoacetamide for 1 h in the dark, and desalted on C 18 reversed-phase columns (Millipore) with a stepwise elution with 20, 40, 60, and 80% acetonitrile in 0.1% trifluoroacetic acid.…”

Mouse AMACO, a kidney and skin basement membrane associated molecule that mediates RGD-dependent cell attachment

“…AMACO is a recently discovered protein containing von Willebrand (VWA) domains related to those found in matrilins and collagens (Sengle et al, 2003). The mature secreted protein has a calculated M r of 83,024.…”