Calreticulins- Calcium-Binding Proteins with Functional Diversity and Evolutionary Duality

University dissertation from Department of Biochemistry (S), Lund University

Abstract: Calreticulin is a multifunctional protein mainly localized in the endoplasmic reticulum in eukaryotic organisms, except yeasts. The protein comprises three distinct regions: the N-terminal N domain, the C-terminal C domain, and the central P domain, reflecting the functional diversity of calreticulin. The protein has been implicated in over 40 intra- and extracellular processes in mammalian cells, but the main research focus has been on its role in protein folding and as a regulator of cellular calcium homeostasis. The protein can bind approximately 25 mol Ca2+ per mol protein, largely mediated by the negatively charged C domain. Although a similar Ca2+ binding capacity is evident for plant calreticulins, the physiological relevance of the protein in endoplasmic reticulum Ca2+ regulation has been elusive. Nicotiana tabacum suspension cells were therefore transformed with a Zea mays crt1a cDNA, under the control of a heat-shock promoter. Induction of the calreticulin transgene enhanced the endoplasmic reticulum Ca2+ content in vitro. Furthermore, heat shock-induced Arabidopsis plants, transformed with the same construct, grew better on medium containing low levels of Ca2+ compared with control plants. The multifunctional properties assigned to calreticulins have triggered a search for additional isoforms and for two or more copies of calreticulin genes in mammals. By performing BLASTP searches we found a calreticulin isoform (Crt2) in human, which differed significantly from the previously established isoform. In addition, a homolog to the Crt2 protein was obtained from mouse, and crt2-related ESTs were found in rat and pig. The data thus supported an orthologous calreticulin isoform present in several mammalian species. Similar to the calnexin homolog calmegin, the crt2 gene was exclusively expressed in testis of the tissues investigated. Arabidopsis contains three calreticulin isoforms. Phylogenetic analyses and expression profiling revealed that both monocotyledons and eudicotyledons contain two distinct calreticulin isoform groups: a Crt1/Crt2 and a Crt3 group. Whereas the crt1/crt2 genes were active in all tissue types investigated, peaking in flowers, the crt3 gene was mainly expressed in root and leaf tissues. Furthermore, members from the different isoform groups were induced differently in response to tunicamycin, an inhibitor of N-linked glycosylation of newly synthesized proteins. To provide a common research basis for plant calreticulins, a new nomenclature for the proteins was suggested. The discovery of two orthologous calreticulin isoform groups in both animals and plants support an evolutionary duality, and suggests functional diversity for calreticulins.

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