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

Gerhard Sengle,Birgit Kobbe,Matthias Mörgelin,Mats Paulsson,Raimund Wagener

doi:10.1074/jbc.m307794200

Gerhard Sengle, Birgit Kobbe + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m307794200

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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.

Highlights

The genes coding for human and mouse AMACO, an extracellular matrix protein containing von Willebrand factor A (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 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 mouse AMACO cDNA of 2653 bp contains an open reading frame of 2372 bp, encoding a protein consisting of 791 amino acids residues, preceded by a signal peptide of 23 residues as predicted by a method using neural networks [24]

Summary

EXPERIMENTAL PROCEDURES

RT-PCR and 5Ј RACE—RT-PCR was used to clone the mouse and human AMACO cDNAs. Primers were designed according to EST and genomic sequences (Table I). After filtration and centrifugation (1 h, 10,000 ϫ g), the AMACO-containing cell culture supernatant was concentrated on a DEAE-Sepharose Fast Flow column (2 ml, Amersham Biosciences), and proteins were eluted with 1 M NaCl, 10 mM Tris-HCl, pH 8.0. The eluate containing the C-terminally strepII-tagged AMACO was applied to a streptactin column (1.5 ml, IBA GmbH) and eluted with 2.5 mM desthiobiotin, 10 mM Tris-HCl, pH 8.0. 1— continued for 1 h with 1% (w/v) bovine serum albumin in TBS and incubated with the affinity-purified AMACO antibody overnight at 4 °C. Immunolabeling was done by incubation with affinity-purified antibodies to AMACO for 1 h, followed by CyTM3-conjugated affinity-pure donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Electron Microscopy—Negative staining was performed as described previously [22, 23], using non-reduced samples of the C-terminally strepII-tagged AMACO (5–10 ␮g/ml) stained with 0.75% uranyl formate

RESULTS

I I I 0 0 II II II II I I II II IIIIIIIIII

DISCUSSION