Guinea pig S19 ribosomal protein as precursor of C5a receptor-directed monocyte-selective leukocyte chemotactic factor.

OBJECTIVE: To reveal the C5a receptor-mediated monocyte-selective chemoattraction of the homo-dimer of guinea pig S19 ribosomal protein (RP S19), and to study the topological relationship between the RP S19 and C5a receptor genes. METHODS: cDNA cloning and nucleotide sequencing, leukocyte chemotaxis measurement, and fluorescent in situ hybridization (FISH) were performed in the guinea pig. RESULTS: The amino acid sequence of the guinea pig RP S19 deduced from the cDNA nucleotide sequence was identical to the human protein. The dimer of a recombinant RP S19 attracted guinea pig monocytes but suppressed neutrophil chemotactic movement. Both effects were C5a receptor-mediated. In the FISH analysis, the signals denoting the guinea pig RP S19 gene and C5a receptor gene completely overlapped each other. CONCLUSIONS: The guinea pig RP S19 dimer possessed a dual ligand effect, agonistic to the monocyte C5a receptor and antagonistic to the neutrophil receptor. The RP S19 and C5a receptor genes co-localized on the same chromosome.

Whale high-molecular-weight and low-molecular-weight kininogens.

The expression of high-molecular-weight and low-molecular-weight kininogen mRNAs in the whale liver was examined by reverse transcription-polymerase chain reaction. The nucleotide sequences of the high-molecular-weight and low-molecular-weight kininogen cDNAs were analyzed and deduced to the amino acid sequences. The high-molecular-weight kininogen composed of 609 amino acid residues with 18 signal peptides possessed the consensus sequences of the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. Except for the histidine-rich region, the overall homologies with bovine, human, and rat high-molecular-weight kininogens were 81%, 76%, and 62%, respectively. The low-molecular-weight kininogen is composed of 408 amino acid residues. The nucleotide sequence down to C(1200) as well as the amino acid sequence till Ile(382) is identical to that of the high-molecular-weight kininogen. The remaining low-molecular-weight kininogen-specific carboxy-terminal portion possessed an amino acid sequence similar to that of the land mammals. The overall homologies with bovine, human, and rat low-molecular-weight kininogens were 82%, 79%, and 64%, respectively. The amino acid sequences of both whale high-molecular-weight and low-molecular-weight kininogens are most similar to those of the bovine among the land mammals analyzed so far. An incubation of dolphin/whale plasma with human plasma kallikrein, or with bovine trypsin, in the presence of carboxypeptidase inhibitors generated bradykinin antigen as well as the spasmogenic activity to the estrous rat uterus. The amount of bradykinin released by the latter enzyme was almost double of the former, indicating that the dolphin/whale plasma contained similar concentrations of low-molecular-weight and high-molecular-weight kininogens.

Primary structure of guinea pig plasma prekallikrein.

A full length guinea pig plasma prekallikrein (PK) cDNA was cloned from a liver cDNA library. The nucleotide sequence with 2242 bp was analyzed and the amino acid sequence with 618 residues was deduced. Kallikrein was purified from guinea pig plasma and cleavage site in the activation was determined. The amino acid sequence around the cleavage site -368Ile-Asp-Ala-Arg-Ile-Val-Gly-375Gly- differed from that of the human PK -368Thr-Ser-Thr-Arg-Ile-Val-Gly-375Gly-. Protease substrates containing penta-peptides which mimicked the sequence of the cleavage sites from P3 to P2' of guinea pig Hageman factor (HF) and PK were synthesized, and kinetic analyses of the hydrolysis by guinea pig activated HF (HFa) and kallikrein were carried out. The combination between HFa and the PK mimicking peptide provided the best kinetics. These results in part explain why the cascade activation of PK by HFa is predominant in the guinea pig system.

Whale Hageman factor (factor XII): prevented production due to pseudogene conversion.

In Southern blot analysis of the Hind III-digested whale genomic DNA obtained from the livers of two individual whales, we detected a single band with a size of five kilobase pairs which hybridized to full length guinea pig Hageman factor cDNA. We amplified two successive segments of the whale Hageman factor gene by polymerase chain reaction (PCR), and sequenced the PCR products with a combined total of 1367 base pairs. Although all of the exon-intron assemblies predicted were identical to those of the human Hageman factor gene, there were two nonsense mutations making stop codons and a single nucleotide insertion causing a reading frame shift. We could not detect any message of the Hageman factor gene expression by northern blot analysis or by reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results suggest that in the whale, production of the Hageman factor protein is prevented due to conversion of its gene to a pseudogene. The deduced amino acid sequence of whale Hageman factor showed the highest homology with the bovine molecule among the land mammals analyzed so far.

Species differences in amino acid sequences of Hageman factor and prekallikrein at region around scissile bond in activation.

The initial step in activation of the plasma kinin system is activation of Hageman factor (coagulation factor XII) and/or plasma prekallikrein. Two types of activation mechanisms, contact activation on a negatively charged surface and a cascade activation by exogenous proteases are known. Since these factors are serine protease zymogens, the activation of these molecules usually results from the cleavage of a particular -Arg-Ile(Val)- bond in either mechanism. Hence, these zymogens are regard to be substrates of their activator proteases. Sensitivity of the substrate for the protease basically depends on the amino acid sequence of six to eight residues around the scissile bond of the substrate. We found different activation efficiencies of these zymogens between human and guinea pig in both types of activation, and micro-heterogeneity of the sequence around the scissile bond among human, guinea pig and bovine Hageman factors, or between human and guinea pig prekallikreins. The sequence heterogeneity may explain the different activation efficiencies of these zymogens among mammalian species.

Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules.

A bovine Hageman factor cDNA was cloned from a liver cDNA library. The nucleotide sequence was analyzed and the amino-acid sequence was deduced. The sequence deduced was consistent with the partial amino-acid sequences of bovine Hageman factor protein. The sequences for three portions including the amino terminal had been previously reported (Fujikawa et al. (1977) Biochemistry 16, 2270-2278). In comparison with the primary structures of human and guinea pig Hageman factors, the putative domain structures were totally conserved. Each domain possessed high sequence homology with the human molecule (66-88%) and the guinea pig one (63-81%) except for the proline-rich region (less than 10%) which connects the amino-terminal five domains with a serine proteinase portion. Significant heterogeneities were observed among the three species around the essential cleavage sites for the conversion to the activated Hageman factors. Bovine Hageman factor has no suitable amino-acid sequence as the substrate for the trypsin-type proteinases at the proline-rich region in difference from the human and guinea pig molecules. Probably this is the reason why the beta-form activated Hageman factor (the proteinase moiety) is not liberated in the activation of the bovine molecule with trypsin or plasma kallikrein.

Difference between human and guinea pig Hageman factors in activation by bacterial proteinases: cleavage site shift due to local amino acid substitutions may determine the activation efficiency of serine proteinase zymogens.

Human and guinea pig Hageman factors have been subjected to the action of pseudomonal elastase and serratial E15 proteinase. The pseudomonal elastase cleaved 22-24% of the human molecule at Arg353-Val354, and the remainder at Gly357-Leu358 resulting in the generation of about 20% of potential activity as activated Hageman factor, compared with trypsin activation, while it hydrolyzed Arg340-Ile341 bond in guinea pig molecule and generated about 75% of activity as activated Hageman factor. The serratial proteinase did not hydrolyze the essential cleavage site (Arg353-Val354) of the human zymogen but Gly356-Gly357 (30%) and Gly357-Leu358 (70%) bonds. Both products showed no activity. The guinea pig zymogen, in contrast, was cleaved mostly at Arg340-Ile341 (70%) and less abundantly at Gly344-Leu345 (30%), generating about 85% of the whole potential activity as activated Hageman factor. From the high correspondence between the proportions of activation and of hydrolysis at the essential cleavage site in activation, it was concluded that hydrolysis of the bonds different from the essential bond did not cause activation, even when the spatial separation was only 3 or 4 residues. Considering the amino acid differences between human and guinea pig Hageman factors, -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val-Ala360- and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val-Ala347-, respectively, it was realized that even the minor amino acid substitutions caused the cleavage site shift which resulted in significant differences in activation efficiency of the proteinase zymogens.

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured. The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 degrees C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.

Induction of interferon-gamma production by human natural killer cells stimulated by hydrogen peroxide.

Interferon (IFN)-inducing activity of hydrogen peroxide in human peripheral mononuclear cells was investigated. Among the mononuclear cells, purified nonadherent cells produced IFN, but not B cells and monocytes. The maximal titer of IFN by purified nonadherent cells was observed after a 72-hr cultivation in the presence of 10(-2) mM H2O2 without affecting their viability. Furthermore, the purified nonadherent cells, but not the unpurified mononuclear cells, showed an augmented cytotoxicity to K562 when stimulated with hydrogen peroxide. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into the low and high density fractions for which natural killer (NK) cells and T cells were enriched, respectively. The NK-enriched low density fractions, but not the T cell-enriched high density fractions, showed IFN production by the stimulation of hydrogen peroxide. IFN production as well as large granular lymphocytes and HNK-1+, Leu-11+ cells of the NK-enriched fractions were abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1+) but not against T cells (OKT3) in the presence of complement. Moreover, hydrogen peroxide-inducing IFN production seems to be regulated by monocytes. The antiserum neutralizing IFN-alpha and IFN-beta failed to neutralize substantially IFN-produced NK cells. The treatment with either pH 2 or antiserum-neutralizing human IFN-gamma resulted in marked reduction, indicating that a major part of IFN was IFN-gamma. The purified nonadherent cells showed IFN production and augmented cytotoxicity when cultured separately from activated macrophages by opsonized zymosan; furthermore, both IFN production and enhancement of cytotoxicity were abrogated by catalase. These results suggest that both exogenous and endogenous hydrogen peroxide might be responsible for a part of immunoregulation.