Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and mu-types. Specific expression of the mRNA in skeletal muscle

Two types of calcium-dependent protease with distinct calcium requirements (termed muCANP and mCANP) are known in mammalian tissues. These two isozymes consist of different large (80-kDa) subunits (mu- or m-types) and identical small (30-kDa) subunits. By screening human and rat muscle cDNA libraries with a cDNA probe for the chicken CANP large subunit, which has a structure similar to both the mammalian mu- and m-types, a cDNA clone encoding a novel member of the CANP large subunit family was obtained. The encoded protein (designated "p94") consists of 821 amino acid residues (Mr 94,084) and shows significant sequence homology with both human mu-type (54%) and m-type (51%) large subunits. p94 can be divided into four domains (I-IV) as reported for the CANP large subunit family. Domains II and IV are potential cysteine protease and calcium-binding domains, respectively, and have sequences homologous to the corresponding domains of other CANP large subunits. However, domain I of p94 is significantly different from others. Moreover, p94 contains two unique sequences of 62 and 77 residues in domains II and III, respectively. In contrast to the ubiquitous expression of mu- and m-types, Northern blot analysis revealed that the mRNA for p94 exists only in skeletal muscle with none detected in other tissues including heart muscle and smooth muscles such as intestine.     

Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

Molecular cloning of the cDNA for the large subunit of the high-Ca2+-requiring form of human Ca2+-activated neutral protease

A nearly full-length cDNA clone for the large subunit of high-Ca2+-requiring Ca2+-activated neutral protease (mCANP) from human tissues has been isolated. The deduced protein, determined for the first time as an mCANP, has essentially the same structural features as those revealed previously for the large subunits of the low-Ca2+-requiring form (muCANP) [Aoki, K., Imajoh, S., Ohno, S., Emori, Y., Koike, M., Kosaki, G., & Suzuki, K. (1986) FEBS Lett. 205, 313-317] and chicken CANP [Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M., & Suzuki, K. (1984) Nature (London) 312, 566-570]. Namely, the protein, comprising 700 amino acid residues, is characterized by four domains, containing a cysteine protease like domain and a Ca2+-binding domain. The overall amino acid sequence similarities of the mCANP large subunit with those of human muCANP and chicken CANP are 62% and 66%, respectively. These values are slightly lower than that observed between muCANP and chicken CANP (70%). Local sequence similarities vary with the domain, 73-78% in the cysteine protease like domain and 48-65% in the Ca2+-binding domain. These results suggest that CANPs with different Ca2+ sensitivities share a common evolutionary origin and that their regulatory mechanisms are similar except for the Ca2+ concentrations required for activation.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

E-F hand structure-domain of calcium-activated neutral protease (CANP) can bind Ca2+ ions

The cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18). The resulting plasmids were used to transform E. coli, and isopropyl-1-thio-beta-D-galactoside (IPTG)-inducible expression was performed. The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay. Most of the E-F hand structures could bind calcium and 2 or 4 mol of Ca2+ ions bound to the four consecutive E-F hand structures. The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.     

Primary structure and evolution of calcium-activated neutral protease (CANP)

The amino acid sequences of two subunits (80K and 30K) of calcium-activated neutral protease (CANP) were examined to clarify the structure-function relationship of CANP. The 80K subunit is composed of four clear domains (I–IV from the N-terminus). Domain II is a cysteine proteinase domain homologous to cathepsins B, L, and H. Domain IV is a calcium binding domain with four consecutive EF-hand structures known as typical calcium-binding sites found in calmodulin. The 30K subunit also has a clear domain structure (two domains). The N-terminal domain, a Gly-rich hydrophobic domain, probably determines the location of CANP through association with cellular membrane. The C-terminal domain is a calmodulinlike calcium-binding domain highly homologous to IV in the 80K subunit. The protease activity ascribable to II is regulated by 2 moles of built-in calmodulins, though its precise regulation mechanism is unknown. These results are discussed together with the molecular evolution of CANP on the basis of the gene structures of the two subunits.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Integrin LFA-1 alpha subunit contains an ICAM-1 binding site in domains V and VI.

In order to identify a binding site for ligand intercellular adhesion molecule-1 (ICAM-1) on the beta 2 integrin lymphocyte function-associated antigen-1 (LFA-1), protein fragments of LFA-1 were made by in vitro translation of a series of constructs which featured domain-sized deletions starting from the N-terminus of the alpha subunit of LFA-1. Monoclonal antibodies and ICAM-1 were tested for their ability to bind to these protein fragments. Results show that the putative divalent cation binding domains V and VI contain an ICAM-1 binding site. A series of consecutive peptides covering these domains indicated two discontinuous areas as specific contact sites: residues 458-467 in domain V and residues 497-516 in domain VI. A three-dimensional model of these domains of LFA-1 was constructed based on the sequence similarity to known EF hands. The two regions critical for the interaction of LFA-1 with ICAM-1 lie adjacent to each other, the first next to the non-functional EF hand in domain V and the second coinciding with the potential divalent cation binding loop in domain VI. The binding of ICAM-1 with the domain V and VI region in solution was not sensitive to divalent cation chelation. In short, a critical motif for ICAM-1 binding to the alpha subunit of LFA-1 is shared between two regions of domains V and VI.     

Carboxyl-terminal truncation and site-directed mutagenesis of the EF hand structure-domain of the small subunit of rabbit calcium-dependent protease

A mutant of the small subunit of rabbit calcium-dependent protease lacking the amino-terminal one-fourth produced in Escherichia coli could associate with the native large subunit to exert protease activity. Deletion of a few carboxyl-terminal residues of this variant small subunit caused a significant decrease in the protease activity after reconstitution with the native large subunit. Loss of the fourth EF hand loop region by further truncation of the variant small subunit made interaction with the large subunit impossible. The calcium binding assay revealed that the fourth EF hand structure of the rabbit small subunit, which has been previously demonstrated to possess two calcium-binding sites, can bind calcium ions. Furthermore it was established by site-directed mutagenesis that the first EF hand structure, in addition to the fourth one, is capable of binding calcium ions. Replacement of amino acids in the EF hand structure affected interaction with the native large subunit or the calcium sensitivity of the reconstituted product. These findings indicate that the EF hand structure-domain of the small subunit is essential for the full protease activity.     

An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen

A Mr 23,000 Ag of the human trematode parasite, Schistosoma mansoni, has been identified by immunoscreening an adult worm cDNA library with antibody affinity purified on the Mr 23,000 to 25,000 integral membrane protein fraction of the parasite. This Ag is immunogenic in infected humans as well as in rabbits exposed to S. mansoni. The protein sequence of the Ag as deduced from cloned DNA sequences is 218 amino acids long and contains four putative transmembrane regions. Of particular significance, the Ag is strikingly similar, with respect to both amino acid sequence (36% identity) and putative domain structure to ME491, a human stage-specific melanoma-associated Ag.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli

We have already determined the primary structure of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) from the cDNA sequence and revealed that the CANP inhibitor contains four internally repeating units which could be responsible for its multiple reactive sites (Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3590-3594). Restriction fragments of the cDNA corresponding to each of the four domains (encoding 104-156 amino acid residues of the total 718 residues) were subcloned into the multicloning site of pUC9 or pUC18 in a direction and frame matched to the lacZ' open reading frame of the vector. Under the lac operator-promoter system, we succeeded in producing truncated fragments of the CANP inhibitor in Escherichia coli. The CANP inhibitor fragments were partially purified, and the inhibitory activities toward calcium-dependent protease (CANP) were examined. All fragments containing well conserved regions of about 30 amino acid residues (domains I-IV) located in the middle of the four units exhibited the inhibitory activity. However, their inhibitory activities varied considerably. Further truncation experiments revealed that small fragments containing 30-70 amino acid residues of the CANP inhibitor still retained inhibitory activity. From these experimental results the following conclusions can be drawn: 1) each of the four repeating units of the CANP inhibitor (about 140 amino acid residues) is a real functional unit and can inhibit CANP activity independently; and 2) domains corresponding to well conserved sequences of about 30 amino acid residues containing a consensus Thr-Ile-Pro-Pro-X-Tyr-Arg sequence are essential for the inhibitory activity, and the bordering regions are important for its modulation.     

NADPH Oxidase Genes from Tomato (Lycopersicon esculentum) and Curly-Leaf Pondweed (Potamogeton crispus)

Abstract: The oxidative burst is an integral component of plant resistance to pathogens. There is accumulating evidence that the oxidative burst is catalyzed by an enzyme with similarities to the phagocyte NADPH oxidase. We have cloned a full length homolog of the gp91 ( phox ) subunit of the plasma membrane NADPH oxidase complex from tomato named LeRBOM. The predicted protein contains 989 amino acids. The large N-terminal domain contains two EF hand calcium binding motifs and one conserved glycosylation site. Six putative membrane spans are present in the C-terminal half of the predicted protein. Extensive homology with the human gp91 ( phox ) subunit was found including conservation of amino acid residues important for heme coordination and substrate binding. We have also isolated partial genomic clones from tomato and from the aquatic plant Potamogeton crispus. These species serve as models for studies of signal transduction leading to NADPH oxidase activation. In tomato, LeRBOH1 expression was too low to be detected on Northern blots. RT-PCR indicated that LeRBOH1 was expressed in all tissues tested.     

Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo

The Saccharomyces cerevisiae SEC65 gene encodes a 32 kDa subunit of yeast signal recognition particle that is homologous to human SRP19. Sequence comparisons suggest that the yeast protein comprises three distinct domains. The central domain (residues 98–171) exhibits substantial sequence similarity to the 144 residue SRP19. In contrast, the N-terminal and C-terminal domains (residues 1–97 and 172–273 respectively) share no similarity to SRP19, with the exception of a cluster of positively charged residues at the extreme C-terminus of both proteins. Here, we report the cloning of a Sec65p homologue from the yeast Candida albicans that shares the same extended domain structure as its S. cerevisiae counterpart. This conservation of sequence is reflected at the functional level, as the C. albicans gene can complement the conditional lethal sec65-1 mutation in S. cerevisiae . In order to examine the role of the N- and C- terminal domains in Sec65p function, we have engineered truncation mutants of S. cerevisiae SEC65 and tested these for complementing activity in vivo and for SRP integrity in vitro . These studies indicate that a minimal Sec65p comprising residues 76–209, which includes the entire central SRP19-like domain, is sufficient for SRP function in yeast.     

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.     

Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.     

Rat brain calbindin D28: six domain structure and extensive amino acid homology with chicken calbindin D28

Calbindin D28 cDNA clones were isolated from a rat brain library using a chicken intestinal Calbindin D28 cDNA probe. Nucleotide sequence analysis of these clones shows an open reading frame of 78 nucleotide coding for a 261 amino acid 29,994 dalton protein. The predicted amino acid sequence contains six repeats of a domain with the feature of an EF-hand calcium binding site. In domains II and VI, two of the five oxygen-containing amino acids important for the coordination of calcium are absent, suggesting that these two sites have lost their calcium-binding capability. Comparing the amino acid sequence to that recently reported for the chicken Calbindin D28 there is 79% homology. Tolerating conservative differences, the homology increases to 93%. Interestingly, domains II and VI which have presumably lost their calcium binding ability are very conserved among the two species (81% and 78%, respectively). Since an EF hand calcium binding site requires only certain types of amino acids at certain positions, rather than a specific amino acid sequence, maintaining a calcium binding site is a weak conservation pressure. To explain the high degree of homology of rat and chicken Calbindin D28, and in particular the conservation of the two degenerated domains over the 300 million years since divergence of birds and mammals, additional function(s) of the Calbindin D28 are postulated.     

Streptococcal-host interactions. Structural and functional analysis of a Streptococcus sanguis receptor for a human salivary glycoprotein

Colonization of oral tissues by Streptococcus sanguis may be influenced by a mucin-like salivary glycoprotein (SAG) through a calcium-dependent interaction with a specific bacterial receptor. We report the nucleotide and deduced amino acid sequence of the S. sanguis receptor (SSP-5) and show that this protein may bind sialic acid residues of SAG. The SSP-5 protein contains three unique structural domains, two of which consist of repetitive amino acid sequences. The N-terminal domain is comprised of four tandem copies of an 82-residue repeat which exhibits homology to M protein of Streptococcus pyogenes. This region is highly charged and predicted to be alpha-helical. A second hydrophilic repetitive domain consists of three copies of a 39-amino acid sequence containing 30% proline flanked by nonrepetitive proline-rich sequence. The third domain consists of 48% proline and resides near the C terminus of the protein. Secondary structure analysis of the SSP-5 sequence also identified four potential helix-turn-helix motifs that resembled E-F hand calcium binding domains. The SSP-5 protein is highly homologous to a surface antigen expressed by the mutans streptococci and the domain structure of SSP-5 is conserved within this family of proteins. The interactions of SSP-5 and of intact S. sanguis with SAG were inhibited by neuraminidase digestion of the salivary glycoprotein and by simple sugars containing sialic acid, suggesting that sialic acid is the primary ligand involved in the binding reaction.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.