Isolation of two isoforms of a novel 15-kDa protein from rabbit polymorphonuclear leukocytes that modulate the antibacterial actions of other leukocyte proteins

We have recently reported the use of the highly selective and reversible binding of the potent bactericidal/permeability-increasing protein (BPI) to target Gram-negative bacteria (Escherichia coli) for its isolation from crude extracts of human polymorphonuclear leukocytes (PMN). We now report the use of the same procedure for the purification from rabbit PMN of BPI and also of a novel 15-kDa species that consists of two nearly identical isoforms. These 15-kDa proteins have no demonstrable antibacterial activities by themselves. However, one isoform (p15A) potentiates strongly and the other (p15B) weakly the early antibacterial effects of both rabbit and human BPI. Both isoforms inhibit the late lethal action of BPI. Whereas the potentiating effect is specific for BPI the inhibitory effect is seen also with another antibacterial protein of PMN granules, azurocidin. Thus, we have identified in rabbit PMN a previously unrecognized 15-kDa protein species that may modulate during phagocytosis the antimicrobial effects of BPI (and other granule proteins).     

Relation between binding and the action of phospholipases A2 on Escherichia coli exposed to the bactericidal/permeability-increasing protein of neutrophils

Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A2 to E. coli in the presence and absence of BPI. Phospholipases A2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH2-terminal amino sequences of the active and inactive phospholipase A2 suggests that this portion of the phospholipase A2 molecule plays a role in BPI-independent binding and hydrolysis.     

Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.     

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.     

Domain structure of the HSC70 cochaperone, HIP.

The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.     

The BPI/LBP family of proteins: a structural analysis of conserved regions.

Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), share high-affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of gram-negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side-chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid-binding pockets. Most of the conserved polar and charged residues appear to be involved in inter-residue interactions such as H-bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2-terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI''s bactericidal activity is related to the high positive charge of its NH2-terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site-directed mutagenesis and inhibitor design.     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.

The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.     

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

Intracellular cleavage of newly synthesized low affinity Fc epsilon receptor (Fc epsilon R2) provides a second pathway for the generation of the 28-kDa soluble Fc epsilon R2 fragment

It has been reported that the 45-kDa low affinity Fc epsilon R (Fc epsilon R2) on B cells is cleaved spontaneously from the cell surface to release a 28-kDa soluble fragment (sFc epsilon R2). This study demonstrates an additional mechanism by which B cells generate this fragment. Data from 35S methionine pulse-chase experiments with the Fc epsilon R2 bearing human B lymphoblastoid cell line, RPMI 8866, and immunoprecipitations of cell lysates and culture supernatants with an Fc epsilon R2 specific mAb, mAb 25, demonstrates the existence of a cell-associated 28-kDa Fc epsilon R2 fragment. This fragment was shown by partial amino(NH2)-terminal sequence analysis to be identical to the previously described 28-kDa sFc epsilon R2. The resistance to cell treatment with trypsin indicated that it was located intracellularly. Its appearance early in the biosynthesis of the Fc epsilon R2 (within a 10-min pulse), before the Fc epsilon R2 reached the cell surface, suggested that some of this fragment was generated intracellularly. Neutralization of acidic organelles with NH4Cl inhibited the formation of this intracellular fragment, strongly suggesting that it was a produce of intracellular cleavage of the Fc epsilon R2. Finally, this 28-kDa intracellular fragment was shown to be released into the culture supernatant, suggesting an intracellular mechanism by which the cells generate sFc epsilon R2.     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.     

Isolation and molecular and functional properties of the amino-terminal domain of colicin A

A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A.     

The molecular cloning of the gene encoding the Escherichia coli 75-kDa helicase and the determination of its nucleotide sequence and gentic map position

A previously unreported DNA unwinding enzyme, referred to as the 75-kDa helicase, was recently purified from Escherichia coli cell extracts and biochemically characterized (Wood, E. R., and Matson, S. W. (1987) J. Biol. Chem. 262, 15269-15276). In order to initiate the genetic analysis of the 75-kDa helicase, the gene encoding this enzyme was cloned. DNA sequencing confirmed the identity of the gene since the predicted amino acid sequence of the encoded polypeptide precisely matched the sequence of the first 27 NH2-terminal amino acid residues of the 75-kDa helicase as determined by peptide sequencing. The predicted amino acid sequence of the 75-kDa helicase is similar in several regions to the amino acid sequences of two other E. coli helicases, Rep protein and helicase II. The gene encoding the 75-kDa helicase was mapped to 22 min on the E. coli chromosome. We propose that this newly defined locus be referred to as helD, and, to avoid confusion with other E. coli helicases with a similar molecular size, we propose that the 75-kDa helicase be referred to as helicase IV.     

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.     

Identification and properties of the catalytic domain of mammalian DNA polymerase beta

Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.     

The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r) > 20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.     

Myristic acid is the NH2-terminal blocking group of the 43-kDa protein of Torpedo nicotinic post-synaptic membranes

The NH2-terminal blocking group of the 43-kDa peripheral membrane protein (43-kDa protein) of Torpedo post-synaptic membranes has been identified as myristic acid. To identify that blocking group pure 43-kDa protein was digested with trypsin and the blocked tryptic peptide was isolated by reverse phase HPLC. That peptide coeluted with and had the same amino acid composition as a synthetic peptide, myristoyl-Gly-Gln-Asp-Gln-Thr-Lys, the structure of the amino terminus predicted from the protein sequence deduced from a cDNA clone. The presence of myristate was confirmed by the precise molecular mass of the peptide, 886.5266, determined by fast atom bombardment mass spectroscopy.     

Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.