Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.

Abstract

Abstract. The acrosomal process of Limulus sperm is an 80-~m long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin illaments at 13-/~ resolution shows that scruin is organized into tv~ equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organiTation of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Proteasesensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOHterminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an ,,~50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four/~-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

Krp1, a novel kelch related protein that is involved in pseudopod elongation in transformed cells

“…Further kelch proteins are also thought to interact with actin: murine and human ENC1 (Hernandez et al, 1997(Hernandez et al, , 1998; spe-26 of C. elegans (Varkey et al, 1995); and human Mayven (Soltysik-Espanola et al, 1999). Many other family members are thought not to interact with actin; beta-scruin of Limulus, for instance, is found in sperm acrosomal vesicles, which do not contain actin (Way et al, 1995b). In addition, Schizosaccharomyces pombe kelch family member Tea1 is found at the tips of growing cells and is thought to interact with the ends of microtubules rather than with actin (Mata and Nurse, 1997).…”

ENC-1: A Novel MammalianKelch-Related Gene Specifically Expressed in the Nervous System Encodes an Actin-Binding Protein

“…This motif is called the kelch repeat (Bork and Doolittle, 1994;Cooley and Theurkauf, 1994). Among members of this family, kelch, SPE-26, calicin, and ␣-scruin are synthesized as cytoskeletal components during germ cell differentiation, and they occur in membrane-associated dense structures (Xu and Cooley, 1993;Varkey et al, 1995;von Bülow et al, 1995;Way et al, 1995b). Kelch co-localizes with actin filaments that form ring canals that regulate nutrient transport from the nurse cells and oocyte.…”

“…This tandemly repeated motif was first identified in the kelch protein and subsequently defined a family of proteins containing highly similar repeats (Chang-Yeh et al, 1991;Xue and Cooley, 1993;Bork and Doolittle, 1994). Other members of this family that share significant sequence identity with ENC-1 are calicin, a major basic protein of the mammalian sperm head cytoskeleton (von Bülow et al, 1995); SPE-26, a Caenorhabditis elegans protein expressed throughout the testis in both spermatogonial cells and spermatides (Varkey et al, 1995); ␣-scruin, an actin-bundling protein found in the acrosomal process of Limulus polyhemus sperm (Way et al, 1995b); ␤-scruin, a homolog of ␣-scruin that is localized to the acrosomal vesicle of Limulus sperm (Way et al, 1995a); and MIPP, a protein encoded by a intracisternal A-particle-promoted placenta-expressed gene (Chang-Yeh et al, 1991). Additional protein sequences that contain this repeated motif include several ORFs of the poxvirus family such as A55R, C2L, F15, and F3L of vaccinia virus (Goebel et al, 1990); C4L and C13L of swinepox virus (Massung et al, 1993); and P65 of entromelia virus (Senkevich et al, 1993).…”

“…Analysis of ENC-1 indicates that it is a homolog of kelch, a Drosophila gene essential for oogenesis (Xue and Cooley, 1993). Members of the kelch family have been identified in several species and are important for cytoskeletal organization and function (Varkey et al, 1995;Way et al, 1995b). ENC-1 is the only member of this family expressed in the NS and encodes a cytoplasmic protein that interacts with the actin cytoskeleton.…”

Characterization of the Actin Cross-linking Properties of the Scruin-Calmodulin Complex from the Acrosomal Process of Limulus Sperm

“…Based on sequence analysis, limited proteolysis, and EM image reconstructions, scruin is organized into two 40-kDa domains connected by a highly helical protease-sensitive neck (7)(8)(9). Each domain consists of a six-fold repeat of ϳ50 amino acids that based on the work of Bork and Doolittle (10), is predicted to fold into a four stranded ␤-sheet motif (9).…”