Identification and characterization of inhibitory sequences in four repeating domains of the endogenous inhibitor for calcium-dependent protease

We reported previously the cDNA cloning of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli. The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues. The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions. The inhibitory activities of various fragments expressed in E. coli suggest the involvement of the regions around the well-conserved sequences. In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides. The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site. A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none. The inhibitory sequence is suggested as LGXKDREXTIPPXYRXLL. The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor. The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.     

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.     

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.     

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.     

Calcium-activated neutral protease inhibitor from rabbit erythrocytes lacks the N-terminal region of the liver inhibitor but retains three inhibitory units

Endogenous inhibitors for calcium-activated neutral protease (CANP) were purified from rabbit erythrocytes and liver. The purified inhibitors showed single bands but with significantly different mobilities on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Peptide mapping and sequencing analyses have revealed that the erythrocyte inhibitor (429 residues) retains the C-terminal three repetitive units of the liver inhibitor (639 residues), which contains four potential repetitive units for inhibition of CANP. The erythrocyte and liver inhibitors inhibited 3 and 4 moles of CANP on the basis of the molecular weights of 46,000 and 68,000, respectively.     

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 and Expression of cDNA Encoding the Cysteine Proteinase Inhibitor from Upland Cotton

A cDNA encoding a novel cysteine proteinase inhibitor (CPI) was isolated from a gland mutant Xiangmian-18 of upland cotton during the pigments gland forming stage. The cDNA comprises 378 bp and encodes 125 amino acid residues with molecular mass of 13.8 kDa. It contains the conserved motif of cysteine protease inhibitors and belongs to the cystatin superfamily (Gln-Val-Val-Ala-Gly). The deduced amino acid sequences of the domains are highly similar to the normal upland cotton (96.8%). SDS-PAGE and western hybridization analysis showed that the expressed recombinant protein was recombinant CPI. The inhibitory activity of recombinant CPI was 46 u/μg which was measured by inhibiting the protease activity of papain. RT-PCR results indicated that the expression level of developing gland stage was higher than that of undeveloped gland stage.     

Characterization of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni

To identify and characterize Schistosoma mansoni proteins that are recognized by infected hosts, we have used a pool of sera from infected humans to screen cDNA libraries constructed from poly(A)+ mRNA of adult S. mansoni. The deduced amino acid sequences of the three isolated clones showed a high degree of similarity to the large subunit of calcium-activated neutral proteinase (CANP) from humans and chicken. These overlapping clones, which include a nearly full-length clone with an open reading frame of 758 amino acid residues, together encode the entire large subunit of CANP. The deduced sequence of this S. mansoni protein can be divided into four domains (I-IV) that include the two domains characteristic of other large subunits of CANP: a thiol-protease domain (II) and a calcium-binding domain (IV) containing EF hand motifs. However, the schistosome protein is unique in having only three EF hand motifs in the calcium-binding domain and in having an additional EF hand motif that is shared between domains II and III. We have shown that these EF hand motifs are capable of binding 45Ca2+. Furthermore, the large subunit is S. mansoni contains an NH2-terminal sequence of 28 residues that is absent from the mammalian CANPs and has a high degree of similarity to the presumed receptor binding sequence of colicin Ia and Ib.     

Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP) from rabbit skeletal muscle

Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.     

The acid-stable proteinase inhibitor of human mucous secretions (HUSI-I, antileukoprotease). Complete amino acid sequence as revealed by protein and cDNA sequencing and structural homology to whey proteins and Red Sea turtle proteinase inhibitor

The complete amino acid sequence of human antileukoprotease has been determined by direct sequencing of the inhibitory active protein isolated from seminal plasma (HUSI-I) and by sequence analysis of cDNA reverse-transcribed from mRNA prepared from cervical tissue. The inhibitor (Mr 11726) consists of 107 amino acid residues including 16 cysteines presumably forming disulfide bonds. The molecule comprises two consecutive domains which are homologous to each other, to the second domain of the basic protease inhibitor from Red Sea turtle (chelonianin) and to both domains of the whey proteins of rat and mouse. Both domains contain a pattern of cysteines known as the 'four-disulfide-core' that has also been found in wheat germ agglutinin and neurophysin.     

Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity

The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.     

Primary structure of chicken pituitary prolactin deduced from the cDNA sequence. Conserved and specific amino acid residues in the domains of the prolactins

The perform of chicken prolactin (PRL) deduced from the cDNA sequence contains a signal peptide of 30 amino acid residues followed by a mature PRL of 199 residues. Chicken PRL shows 77, 68, 67, 58, and 31% identity of amino acid sequence with whale, human, ovine, rat, and salmon PRLs, respectively. Elucidation of the primary structure of avian PRL enabled extended analysis of the specific and conserved amino acid residues and domains of the PRL molecules. The mammalian, teleostean, and avian PRLs share 32 common residues, and these conserved residues are observed to cluster in four distinct domains (PD1 to PD4), corresponding to four of five conserved domains of the growth hormones. Of the 32 residues, 8 residues in the PD2 and PD4 domains, including 4 cysteines, are conserved by other members of the growth hormone family, which indicates that these 8 residues may be essential for common structural features of the gene family. On the other hand, 13 other residues distributed among all four domains are conserved almost exclusively in the PRLs, suggesting that these residues are indispensable for specific binding of the PRLs to their receptors.     

A unique specificity of a calcium activated neutral protease indicated in histone hydrolysis

Calf thymus histones were found to be susceptible to a calcium-activated neutral protease [CANP: EC 3.4.22.17] which required a high concentration of calcium ions for its activity (mCANP). The susceptibilities of histones were in the order of relative degradation rate: H2B, H2A, and H3. The major peptide fragments released by CANP from H2A, H2B, and H3 were isolated and the cleavage sites were determined. Examination of amino acid sequences and environmental features around the cleavage site as well as kinetic analysis of the degradation process led us to the following conclusions about the mode of substrate recognition of mCANP: 1) The cleavage sites in histones could not be interpreted in terms of the primary structure around them. Thus, it seems unlikely that the specificity of CANP solely depends on its recognition of any specific amino acid residues or sequences. 2) The susceptible bonds were never located in the midst of either a hydrophobic or hydrophilic alignment of amino acid residues but in the vicinity of the boundary between hydrophilic and hydrophobic clusters. 3) Once a peptide fragment was generated by the proteolytic degradation, no further cleavage occurred even if the peptide still contained a bond corresponding to what was susceptible to CANP in an intact histone. This observation was interpreted to mean that CANP may recognize a certain higher order structure of its substrates.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of human milk bile salt activated lipase

The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda gt11 and lambda gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.     

猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析

为了比较猪瘟病毒 (HCV)野毒株、疫苗株及标准株之间E2基因抗原区域的差异 ,采用RT PCR扩增了HCV石门株、兔化弱毒疫苗株、野毒 0 3及 0 7株的囊膜糖蛋白E2 (gp55)全基因的cDNA片段 ,分别克隆于pGEM T载体中并对其进行了核苷酸序列测定及氨基酸序列的推导 ,同时进行了同源性比较及E2结构与功能的分析。所测 4株HCVE2基因的长度均为1 2 73bp,所编码的氨基酸序列均包括部分信号肽序列和完整的跨膜区序列 ,共由 381个氨基酸组成 ;4个毒株E2蛋白N末端的 683位至 690位信号肽序列 (WLLLVTGA)和C末端 1 0 30～1 0 63位跨膜区均为保守序列 ,而且具疏水性 ;N末端抗原功能区中 ,4个E2蛋白与其它所比较序列在位于第 753位至 759位氨基酸处 ,均有一段保守序列RYLASLH ,无一氨基酸发生变异 ,为亲水性 ,在整个E2蛋白抗原谱中抗原性峰值为最高 ,推测对抗原性产生起重要作用 ;4个E2蛋白的氨基酸序列中均含有 1 5个半胱氨酸 (Cys)残基 ,其数量及位置与国外五株HCV(Brescia ,C ,Alfort.ALD和GPE)完全一致。表明…