Isoform Cloning, Actin Binding, and Chromosomal Localization of Human Erythroid Dematin, a Member of the Villin Superfamily

Abstract

Dematin is an actin-bundling protein of the erythroid membrane skeleton and is abundantly expressed in human brain, heart, skeletal, muscle, kidney, and lung. The 48-kDa subunit of dematin contains a headpiece domain which was originally identified in villin, and actin-binding protein of the brush-border cytoskeleton. The head-piece domain of villin is essential for its morphogenic function in vivo. Here we report the primary structure of 52-kDa subunit of dematin which differs from the 48-kDa subunit by a 22-amino-acid insertion within its headpiece domain. A unique feature of the insertion sequence of the 52-kDa subunit is its homology to erythrocyte protein 4.2. The insertion sequence also includes a cysteine residue which may explain the formation of sulfhydryl-linked trimers of dematin. Actin binding measurements using recombinant fusion proteins revealed that each monomer of dematin contains two F-actin binding sites: one in the headpiece domain and the other in the undefined N-terminal domain. Although the actin bundling activity of intact dematin was abolished by phosphorylation, no effect of phosphorylation was observed on the actin binding activity of fusion proteins. Using somatic cell hybrid panels and fluorescence in situ hybridization, the dematin gene was localized on the short arm of chromosome 8. The dematin locus, 8p21.1, is distal to the known locus of human erythroid ankyrin (8p11.2) and may contribute to the etiology of hemolytic anemia in a subset of patients with severe hereditary spherocytosis.

Headpiece domain of dematin is required for the stability of the erythrocyte membrane

“…Our previous biochemical and genetic characterization of dematin has shown that both 48-and 52-kDa subunits of dematin contain a C-terminal headpiece domain that is homologous to the headpiece domain of villin (10,23). The 383-residue, 48-kDa subunit consists of an N-terminal core domain that includes a proline-rich motif (PEST), a polyacidic motif, and a C-terminal 75-residue headpiece domain (23).…”

“…Based on sequence of 48-kDa and 52-kDa subunits, a model has been proposed where the headpiece domain plays a critical role in the assembly of dematin trimer (10). We used a well-established chemical crosslinking approach to test whether the deletion of dematin headpiece domain disrupts the formation of trimer and influences dematin's ability to interact with its surrounding cytoskeletal components (11,24).…”

“…Dematin is a substrate for multiple protein kinases, and phosphorylation of dematin by the cAMP-dependent protein kinase is known to regulate dematin's actin-bundling activity in vitro (7)(8)(9). The major phosphorylation site of the cAMPdependent protein kinase is located within the headpiece domain of dematin (10), but the physiological significance of dematin phosphorylation is not known.…”

“…It is therefore likely that one dematin trimer is associated with one actin oligomer in vivo. Dematin is a trimeric assembly of two 48-kDa polypeptides and one 52-kDa polypeptide (10). The C-terminal 75 residues of both polypeptides are homologous to the headpiece domain of villin, an actin-binding protein of the brush border cytoskeleton (14).…”

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

“…Dematin is a known actin filament bundling protein that is not known to play a role in splicing [47]. The protein, p47 is similar to a zinc finger protein (Zfp462), but the function of this protein is not known (Swissprot database).…”