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Elod Kortvely, Stefanie M. Hauck, Jennifer Behler, Matteo Gorza, Marcel Blindert, Marius Ueffing; Sorting Elements of ARMS2: New Mechanistic Insights into Protein Transport. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2342.
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© ARVO (1962-2015); The Authors (2016-present)
ARMS2 is one of the three genes in close vicinity on chromosomal region 10q26 strongly associated with age-related macular degeneration (AMD). By using immunocytochemical and biochemical analyses, we recently reported that the encoded short (11 kDa) protein is a constituent of the extracellular matrix. Nevertheless, ARMS2 lacks any predictable targeting signal and the mechanism responsible for its secretion has remained puzzling. Therefore we set out to determine the overall trafficking patterns of this intriguing, primate-specific protein.
Cassette and oligonucleotide-directed mutagenesis was carried out to generate a set of plasmids coding for mutated ARMS2. The localizations of the mutated ARMS2 proteins in transfected cells were revealed by immunocytochemistry using our own ARMS2-specific monoclonal antibodies. The export of these mutated proteins was also tested by Western analyses using extra- and intracellular fractions of cultured cells. Immunoprecipitation of ARMS2 was performed in order to identify co-precipitated proteins involved in targeting. A database search identified several proteins possessing the same putative targeting signal identified in ARMS2. A similar in vitro mutagenesis was carried out on a subset of these proteins, and the impact of the introduced mutations was tested with the same experimental regime.
We have previously reported that the isoleucine pair within the last eight amino acids (-SIIHTAAR*) are indispensable for proper targeting of ARMS2. Now we show that this highly hydrophobic motif cannot be substituted with other hydrophobic residues, like valine or leucine. On the other hand, the distance of the di-isoleucine motif and the carboxyl-terminus can be varied without affecting the export of ARMS2. Because the C-terminal di-isoleucine element turned out to be neither transplantable nor autonomous, we sought after additional targeting signals and delineated the region, which plays a crucial role in the secretion of ARMS2.
ARMS2 uses a non-canonical pathway for its secretion via a highly hydrophobic motif shared by only few proteins. Understanding the molecular basis responsible for its sorting may contribute to an understanding of its function, which currently remains elusive.
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