Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis

Proteins of selected embryonic stages were metabolically labeled with [(35)S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.     

Mammalian HSP60 is a major target for an immunosuppressant mizoribine

It has been reported that immunosuppressant cyclosporin A or FK506 binds to immunophilins in the cell and that these immunophilins make a complex with molecular chaperones HSP70 or HSP90. Although mizoribine has been used clinically as an immunosuppressant, immunophilins of the agent have not yet been fully understood. We investigated their specific binding proteins using mizoribine affinity column chromatography and porcine kidney cytosols. By increasing mizoribine in the eluant from the column, two major proteins (with molecular masses of 60 and 43 kDa) were detected by SDS-polyacrylamide gel electrophoresis. Based on the amino acid sequence analysis of these proteins, 60- and 43-kDa mizoribine-binding proteins were identified with HSP60 and cytosolic actin, respectively. A considerable amount of actin was also eluted from the affinity column by nucleotides, but a very low quantity of HSP60 was eluted under the same conditions. On the other hand, HSP60 was eluted as a major protein in the eluant that was eluted preferentially, with nucleotide followed by mizoribine. Actin was also detected in the eluant, but the quantity of the protein was very low. These results indicated that HSP60 has high affinity to mizoribine, and the interaction was also observed on surface plasmon resonance analysis. Although HSP60 or GroE facilitated refolding of citrate synthase in vitro, mizoribine interfered with the chaperone activity of HSP60. On different types of mizoribine affinity columns, HSP60 or actin recognized the NH(2) group of mizoribine, and this group may be a functional group of the agent.     

Developmental changes of heat-shock proteins in porcine testis by a proteomic analysis

Heat-shock proteins (HSPs) are important in spermatogenesis. This study investigated developmental changes in the expression of major HSPs in porcine testis. The testis from five immature (mean age 2.9+/-0.1 months) and five mature boars (35.7+/-14.0 months) were examined. Two-dimensional polyacrylamide gel electrophoresis was conducted and proteins were identified by Western blotting and/or matrix-assisted laser desorption/ionization mass spectrometry. Moreover, the 90, 70, and 60 kDa HSPs, 70 kDa heat-shock cognate protein (HSC 70), tubulin, and actin were quantified on two-dimensional gels. Protein spots were quantified by densitometry, combined with a computer-assisted image analysis system. Immunohistochemistry was performed to analyze the expression pattern of major HSPs and beta-tubulin in testis. One isoform of HSP 90 (HSP 90 alpha), two isoforms of HSC 70 (HSC 70a and HSC 70c), one isoform of HSP70 (HSP 70e), and tubulin increased after sexual maturation (P<0.05). A testis-specific HSP70 (P70t) was markedly increased in the testes of sexually mature boars. Meanwhile, levels of actin and some isoforms of HSPs including 60 kDa HSP remained similar in both groups. These observations were further confirmed by immunohistochemistry; therefore, the upregulation of protein expression in the adult testis could be attributed to a higher level of protein expression and the number of cells that were HSPs-positive already resided in the immature testis. The differential expression of major HSPs suggested that they may be important in porcine spermatogenesis.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.     

Heat Shock Protein Produced by Helicobacter pylori

The cells of Helicobacter pylori were suspended in the medium containing³⁵S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.     

Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration.

There are few reports concerning the sorting mechanisms of mammalian HSP60 into the mitochondria from the cytoplasm. In the present study we investigated the protein import system. Based on immunoblotting and immuno-histochemistry, HSP60 was detected in both the cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay. HSP60 mRNA was increased in the kidney papilla of rats that had been water restricted for three and five days, but no changes in HSP60 mRNA were detected in the cortex or the medulla of the rat kidneys. Upon immunoblotting, HSP60 was detected in both the cytoplasm and the mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the kidney papilla of rats that were water restricted but the protein was increased in the mitochondria of the rat kidney papilla. We also analysed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with a molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from the affinity column by an excess of signal peptide or by 5 mm ATP. Upon immunoblotting, the 70-kDa protein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytoplasmic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP70.     

Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney.

The chaperonin family of proteins, which includes GroEL protein of E. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant chloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial P1 protein. We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA. Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand. After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer. Analysis of column fractions on a 7.5% polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da. Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence. These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support.     

Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

Wuchereria bancrofti: cloning and characterization of heat shock protein 70 from the human lymphatic filarial parasite

Heat shock protein 70 (HSP70) was identified as an immunodominant antigen by screening a Wuchereria bancrofti (Wb) microfilarial cDNA library with pooled Wb-infected sera, with 28% of the immunopositive clones coding for Wb-HSP70. The deduced amino acid sequence showed greater than 97 and 85% identity with HSP70 from filarial nematodes and humans, respectively. Recombinant HSP70 (74 kDa) and a recombinant protein from the C-terminal portion (43 kDa) also reacted with pooled Wb-infected sera, suggesting that the C-terminal region of HSP70 contains at least one antibody epitope. Brugia malayi L3 larvae showed increasing levels of HSP70 with increasing temperatures. Further, a polyclonal mouse anti-Wb-HSP70 antibody had reactivity to the HSP70 of cattle filarial parasite Settaria digitata and to human HSP70 derived from a Hep-2 cell line. Immune reactivity to Wb-HSP70 was strong, with uninfected non-endemic normal sera showing significantly greater reactions than sera from filaria-infected individuals. Both immunodominant self-HSP70 and HSP70 from other microbial infections may be primary targets for developing autoantibodies naturally.     

Characterization of HSP27 and three immunologically related polypeptides during Drosophila development

The low-molecular-weight heat-shock protein HSP27 is made in the absence of heat shock during Drosophila melanogaster development. An analysis of the accumulation of HSP27 during specific stages of development is presented using an antiserum recognizing this protein. Whereas HSP27 is abundant during embryogenesis, the level of this protein begins to decrease in the 20-h old embryo and is no longer detectable in second instar larvae. A high level of HSP27 is again observed in third instar larvae and reaches a maximal level in late pupae. While still abundant in young adult flies of both sexes, a greater amount of HSP27 is found in females with the protein being highly concentrated within the ovaries. Following lysis of whole pupae, about 60% of HSP27 is found in the soluble lysate fraction in a form which sediments between 5 and 20 S. Anti-HSP27 serum also recognizes three other developmentally regulated polypeptides with apparent MW of 33, 85 and 120 kDa. The 33 kDa protein accumulates in pupae while those of 85 and 120 kDa are more abundant in third instar larvae. Unlike HSP27, these proteins are not detected in embryos or ovaries. Immunoblot analysis of V8 proteolytic fragments suggests that HSP 27 and 33 kDa are related polypeptides. Exposure of the developing insect to heat-shock treatment results in increased level of HSP27. In larvae, a small amount of the 33 kDa protein accumulates following heat shock, while in pupae and adult flies a decrease in the concentration of this protein is observed after heat shock. Finally, different cellular localizations and distributions within the pupal body have been found for these developmentally regulated polypeptides.     

A novel influenza A virus activating enzyme from porcine lung: purification and characterization

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.     

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa)

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin

Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa. Heat stress at 42.5 °C appeared to be the optimum temperature for HSP formation in cells grown at 30 °C.The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h.The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli. To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity. There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942. Moreover, the purified HSP64 cross-reacted to anti-E. coli GroEL antibody. To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.     

A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus.

A survey of the heat shock response was carried out in a series of streptomycetes. Four major heat shock proteins (HSPs) were observed in each of four species (Streptomyces albus, S. lividans, S. parvulus, S. viridochromogenes) after pulse labeling with [35S]methionine and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three corresponded to the major procaryotic HSPs Lon, DnaK, and GroEL on the basis of their apparent molecular masses (94 to 100, 70, and 56 to 58 kDa, respectively). In addition, a smaller protein (16 to 18 kDa) was detected in all species but was most dramatically induced in S. albus. Consequently, studies focused on this species. As in other procaryotic systems, thermal induction (elicited by a shift from 30 degrees C to 41 degrees C) of the 70- and 94-kDa proteins was transient and expression returned to uninduced levels after 60 min. In contrast, the 56- to 58-kDa (GroEL) and 18-kDa proteins (HSP18) remained induced for more than 2 h. Two-dimensional gel electrophoresis allowed resolution of at least eight S. albus HSPs. HSP56-58 was composed of multiple acidic protein species, whereas HSP18 appeared to be basic. In spite of these differences in their physical characteristics, the N-terminal peptide sequence of HSP18 was similar to those of GroEL-like proteins found in other organisms and identical to one of the HSP56-58 species. In fact, N-terminal amino acid analysis of the S. albus 56- to 58-kDa species showed that it was composed of two proteins that differed in 3 of 10 positions, an observation that was supported by the detection of two groEL-like genes by Southern hybridization. The amino acid sequence of one of these proteins was identical to that of HSP18. Pulse-chase experiments did not reveal evidence of posttranslational processing of either HSP56-58 or HSP18.