Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin

The comparison of the amino acid sequences of 5 cysteine proteinases: papain, actinidin, rat cathepsins B and H and chicken cathepsin L, demonstrates a striking homology among their sequences. The N-terminal region (residues 1-70 in papain) and C-terminal region (residues 118-212 in papain) display the highest sequence homologies, whereas the lowest sequence homologies are observed in the middle region (residues 71-117 in papain); a segment where most insertions/deletions are observed. The highest sequence homology is observed between rat cathepsin H and chicken cathepsin L. As shown by X-ray studies, papain and actinidin have a clearly defined double domain structure. Each domain contains a core of non-polar side chains, which are retained in cathepsins B, H and L, except for the non-polar residue 203 of the core which is replaced by glutamic acid in cathepsin B. The percentage and the location of alpha-helix and beta-sheets of cathepsins B, H and L, assessed using the methods of Garnier et al. (1978, J. Mol. Biol. 120, 97-120) and Chou and Fasman (1974, Biochemistry 13, 222-245), show that the main ordered structures in papain and actinidin are probably retained in cathepsins B, H and L. The differences observed occur essentially in the middle region, a place where sequences display the lowest homologies and which is far removed from the active site.     

Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.     

Human spleen histone H2B. Isolation and amino acid sequence.

The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and H1, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments I to IV, by limited chymotryptic digestion at pH 5.0 and 15 degrees C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1--40, 41--42, 43--121 and 122--125 of the total sequence. The only new findings were microheterogeneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30% alanine). The sequence of the most basic cluster at residues 27--24, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.     

Antigenicities of stem bromelain. Contribution of three-dimensional structure and individual amino acid residues

The contribution of three-dimensional structure and individual amino acid residues to the antigenicities of macromolecular protein was investigated for a thiol protease stem bromelain as antigen. The extent of the participation was demonstrated by a decrease in antigenicity when the enzyme was denatured in 8 M urea before and after reductive cleavage of intrapeptide disulfide bonds or modified in particular amino acid residues. The results showed that the enzyme treated with 8 M urea without reductive cleavage of disulfide bonds preserved about 90% of antigenicity to antibodies against native stem bromelain, while the enzyme denatured after the reductive cleavage of disulfide bonds brought about almost 80% disappearance of the antigenicity. Modification of individual amino acid side chains revealed that lysine was the most immunodominant amino acid, showing 2.5% contribution per residue, and tyrosine followed with 1.2%. However, acidic amino acids such as flutamic and aspartic acids were found to be as low as 0.3%, and tryptophan was 0.2%. These data suggest that most of the antigenic determinants of stem bromelain are of the steric conformation in which lysine and/or tyrosine are most frequently involved as immunodominant amino acids.     

Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K

Endogenous cysteine proteases were given much attention lately, as their role in a variety of pathophysiological disorders became evident. Amongst them cathepsins, which are thought to be implicated in mediation of osteoporosis, cancer progression, atherosclerosis, and many other conditions, are of considerable interest as drug targets. In the presented work, papain was chosen as a model cysteine protease and panning protocol was optimized for selection of papain-binding phage-displayed peptides from a commercially available combinatorial peptide library. Different selection strategies were applied in order to select high-affinity binders. Ultimately, five cyclic peptides (CNWAAGYNCGGGS-NH2, CWSMMGFQCGGGS-NH2, CWEWGGWHCGGSS-OH, CNWTLGGYKCGGGS-NH2 (all cyclized through formation of intramolecular disulphide bond), and GNWTLGGYKGG (cyclized head-to-tail)) were synthesized and tested for inhibitory activity towards papain and human cathepsins L, B, H, and K. The peptides possess inhibitory constants in the low micromolar to mid-nanomolar range and exhibit certain selectivity for different lysosomal cysteine proteases included in this study.     

Structural studies on the carbohydrate moieties of rat liver cathepsins B and H

Cathepsins B and H from rat liver contain one asparagine-linked sugar chain in each molecule. The sugar chains were liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Paper electrophoresis of the radioactive oligosaccharide fractions revealed that they were mixtures of neutral oligosaccharides only. After fractionation by gel filtration the structure of each oligosaccharide was studied by sequential exoglycosidase digestion in combination with methylation analysis. The sugar chain of cathepsin H was a high mannose type oligosaccharide which varied in size from 5 to 9 mannose residues; on the other hand the major oligosaccharide of cathepsin B was a tetrasaccharide whose structure was Manalpha 1----6Manbeta 1----4GlcNAcbeta 1----4GlcNAc.     

The amino acid sequence of the tryptic peptides from actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis.

The amino acid sequences of the tryptic peptides of the thiol proteinase actinidin from Actinidia chinensis were determined by the manual dansyl--Edman procedure. There are 12 tryptic peptides, which give a polypeptide chain of 220 residues with a mol.wt. of 23500. An alignment of the tryptic peptides was made by using the X-ray-crystallographic data of Baker [(1977) J. Mol. Biol. 115, 263--277] determined at 0.28 nm resolution on crystalline actinidin. Detailed evidence for the amino acid sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50083 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Comparative amino acid sequence analysis of VP4 for VP7 serotype 6 bovine rotavirus strains NCDV, B641, and UK.

In a previous study (S. Zheng, G. N. Woode, D. R. Melendy, and R. F. Ramig, J. Clin. Microbiol. 27:1939-1945, 1989), it was predicted that the VP7 serotype 6 bovine rotavirus strains NCDV and B641 do not share antigenically similar VP4s. In this study, gene 4 and the VP7 gene of B641 were sequenced, and the amino acid sequences were deduced and compared with those of NCDV and bovine rotavirus strain UK. Amino acid sequence homology in VP7 between the three strains was greater than 94%, confirming their relationship as VP7 serotype 6 viruses. VP4 of B641 showed amino acid homology to UK of 94% but only 73% homology to NCDV. Sequence comparison of a variable region of VP8 demonstrated amino acid homology of 53% between B641 and NCDV, whereas B641 and UK were 89% homologous in this region. These results confirm the earlier prediction that although the same serotype by VP7 reactivity, B641 and NCDV represent different VP4 serotypes. This difference in VP4 may have contributed to the lack of homotypic protection observed in calves, implicating VP4 as an important antigen in the active immune response to rotavirus infection in bovines.     

Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L

The normal provision of thyroid hormones to the body requires their release from the prohormone, thyroglobulin (Tg). Previous work established the importance of cathepsins B, D, and L (formerly designated cysteine proteinase I) to this process but had not defined the points of proteolytic attack for each enzyme. In the present study we labeled rabbit Tg in vivo with sodium 125I and performed limited digestions with cathepsins B, D, and L, purified from human thyroids. The resultant peptide fragments were analyzed by amino-terminal sequencing and located within the Tg molecule by comparison with the cDNA-derived sequences from human Tg. We identified three cleavage points for cathepsin B, corresponding to P'1 residues 532, 795, and 2487; four cleavage points for cathepsin L, corresponding to P'1 residues 2389, 2452, 2490, and 2657; and four cleavage points for cathepsin D, corresponding to P'1 residues 551, 1835, 2468, and 2643. None of the cleavage points was near Tgs known hormonogenic sites, but these peptide fragments contained three of the four major hormonogenic sites in rabbit Tg, suggesting some preference for their early proteolytic processing. Cathespin B alone among the three endopeptidases had some exopeptidase activity toward Tg. The cleavage specificities for each of the endopeptidases resembled those described with other protein substrates. Thus, cathepsin D preferentially cleaved bonds between hydrophobic residues, and cathespin L cleaved bonds with hydrophobic residues at P2 and P3. Although cathepsin Bs specificity was less obvious, it produced a major cleavage between 2 leucine residues. The existence of three endopeptidases cleaving at different sites shows that Tg proteolysis is a complex process, suggests synergism among their enzyme activities, and provides a physiological mechanism for selective hormone release, including its regulation by TSH.     

Different immunolocalizations of cathepsins B, H, and L in the liver

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.     

Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences.

The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and alpha-lytic protease have been compared with those of the pancreatic enzymes alpha-chymotrypsin and elastase. This comparison shows that approximately 60% (55-64%) of the alpha-carbon atom positions of the bacterial serine proteases are topologically equivalent to the alpha-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is approximately 84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacteria and pancreatic enzymes without significant primary sequence identity (less than 21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164-182) adopts very different comformations which are associated with their altered substrate specificities.     

Systematic relationships of the ranunculacea based on amino acid sequence data

Plastocyanin partial amino acid sequences (40 residues) of five members of the Ranunculaceae were used, together with many other flowering plant plastocyanin sequences already published, to construct dendrograms. On this basis the Ranunculaceae appear more closely related to the Rosaceae and Fabaceae than to the other families investigated. Dendograms constructed from amino acid sequence data and serological data of five members of the Ranunculaceae were similar.     

Molecular evolutionary analysis based on the amino acid sequence of catalase

Heme-containing catalase sequences from 20 different organisms representing prokaryotes, fungi, animals, and plants have been compiled for phylogenetic reconstruction. Phylogenies based on distance and parsimony analysis show that fungal and animal catalases can be derived from one ancestor, whereas bacterial catalases fail to form a monophyletic group. Plant catalases appear to form a second class of catalases that arose independently from a possible prokaryotic ancestor.Correspondence to: P.C. Loewen     

Human spleen histone H1. Isolation and amino acid sequence of a main variant, H1b

The complete amino acid sequence of a main variant, H1b, of human spleen histone H1 was determined, following previous determinations of human spleen histones H2B, H2A, H3, and H4. High-performance liquid chromatography on C8 silica of the H1 fraction yielded the homogeneous H1b subfraction; this variant was estimated to account for 60% of the total of the four H1 variants. The sequence determination was performed with four main fragments, I to IV, obtained by limited chymotryptic digestion of H1b. Together with direct sequencing by automated Edman degradation of fragments II, III, and IV, fragment I, blocked at the N-terminal, and fragment IV, the C-terminal half the H1b molecule, were sequenced after further digestion with staphylococcal protease and others. The four fragments were aligned with three overlapping peptides each derived from chymotryptic partial fragments, I-II and I-II-III, and intact H1b. Carboxypeptidase digestion of intact H1b confirmed the C-terminal sequence of the molecule. Thus, the total sequence of H1b was completely determined; it consists of a total of 218 amino acid residues, has a molecular weight of 21,734 in the unmodified form, and is completely acetylated at the N-terminal serine residue and partially methylated at the lysine residue 25. This sequence is compared with two mammalian somatic H1 sequences.     

Studies on the hydrazinolysis of glycoproteins. Core structures of oligosaccharides obtained from Porcine thyroglobulin and pineapple stem bromelain.

Hydrazinolysis of porcine thyroglobulin glycopeptides and of pineapple stem bromelain [EC 3.4.22.4] permitted the isolation of almost intact carbohydrate chains of these glycoproteins. On the basis of permethylation analyses of the released oligosaccharides after reduction with NaBH4, the core structures of Unit A-type and Unit B-type carbohydrate chains of porcine thyroglobulin were deduced to be Manalpha1 leads to 6[Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[Ralpha1 leads to 6]GlcNAc leads to Asn (Unit A-type, R=H; Unit B-type, R=Fuc), and that of bromelain was found to be Manalpha1 leads to 6[R'1 leads to 2]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[R1 leads to 3]GlcNAc leads to Asn (R'=Xylbeta and R=Fucalpha, or R'=Fucalpha and R=Xylbeta). From these results, it appears that the hydrazinolysis method is applicable to wide variety of glycoproteins which have an N-glycosylamine linkage between the carbohydrate and peptide moieties, regardless of the type of linkage to the most proximal N-acetylglucosamine residue which is bound to asparagine.     

The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin.

Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.     

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.