Identification of a gene required for maturation of an extracellular lactococcal serine proteinase.

Directly upstream of the Lactococcus lactis subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORF1 was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking ORF1 were phenotypically proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.     

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.     

Assembly of the OmpF porin of Escherichia coli B. Immunological and kinetic studies of the integration pathway

The different conformations of the outer membrane protein OmpF of Escherichia coli B were studied with immunological probes. The antigenic determinants recognized by one monoclonal (MoF3) and two polyclonal antibodies were investigated under various conditions of solubilization which modify the association of OmpF with other membrane components, such as lipopolysaccharide. Several polymeric forms of the protein could be detected after extraction at 37 degrees C or 56 degrees C. The monoclonal antibody, which is specific to an exposed region of native OmpF, recognized various trimeric forms in an immunoprecipitation assay. Under the same conditions, the binding of polyclonal antibodies apparently induced strong conformational rearrangements, since the pattern of trimeric forms detected was greatly modified. The conversion of newly synthesized monomers of OmpF to the various trimer forms was investigated using these antibodies. The trimerization occurred rapidly but the appearance of the native conformation of OmpF was delayed. Some additional step was required to expose the MoF3-specific antigenic site at the surface of the trimeric form. These results are discussed in relation to the structure of OmpF and its association with lipopolysaccharide in the outer membrane.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides.

The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.     

The use of monoclonal antibodies to distinguish several chemically modified forms of human alpha 1-proteinase inhibitor.

The purpose of our investigation was to obtain monoclonal antibodies that could distinguish three forms of alpha 1-proteinase inhibitor (alpha 1-PI): native alpha 1-PI, N-chlorosuccinimide-oxidized alpha 1-PI (Ox-alpha 1-PI) and proteolytically modified alpha 1-PI (alpha 1-PI). Three specific monoclonal antibodies were characterized as to their binding properties. By using the Bio-Dot assay, it was found that all three forms of alpha 1-PI were capable of binding to antibody 6D4-6-18, that only Ox-alpha 1-PI, but not native alpha 1-PI or alpha 1-PI, could bind to antibody 6C7-5, and that alpha 1-PI and a complex between alpha 1-PI and trypsin uniquely were not able to bind to antibody 5C12-8-7. Thus it was concluded that it is possible to use monoclonal antibodies with different epitopic specificities to distinguish two chemically modified forms of alpha 1-PI from the native protein.     

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.     

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.     

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies

Summary SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG_1, isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1–126 and 49–149 but failed to bind to fragment 99–149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.     

Proteins of the sea urchin egg vitelline layer

The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.     

Monoclonal antibodies against distinct determinants of histone H5 bind to chromatin

A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.     

A solid-phase competitive radioimmunoassay for the insulin receptor

A radioimmunoassay for the insulin receptor has been developed. In this assay, unlabeled receptor competes with 125I-labeled receptor for binding to monoclonal anti-receptor antibodies immobilized on microtiter wells coated with affinity-purified anti-mouse immunoglobulin G. This assay was highly reproducible and could detect 7 ng (14 fmol) of insulin receptor. By utilizing monoclonal antibodies to various antigenic regions of the receptor, different parts of the receptor molecule could be examined. By utilizing antibodies to the cytoplasmic domain of the receptor, an assay was developed which was not influenced by the presence of insulin and could equally detect the insulin receptor from different species (rat and human) and different tissues (placenta and brain). By utilizing antibodies to an autophosphorylation site of the receptor, the assay was shown capable of detecting the extent of phosphorylation of the receptor. Finally, this assay was utilized to monitor the decrease in insulin receptors in lysates of insulin-treated human lymphocytes. This radioimmunoassay should be useful for monitoring both the number and status of the insulin receptor under a variety of physiological conditions.     

Dissecting the Alternatively Folded State of the Antibody Fab Fragment

Intact antibodies and antigen binding fragments (Fab) have been previously shown to form an alternatively folded state (AFS) at low pH. This state consists primarily of secondary structure interactions, with reduced tertiary structure content. The AFS can be distinguished from the molten globule state by the formation of nonnative structure and, in particular, its high stability. In this study, the isolated domains of the MAK33 (murine monoclonal antibody of the subtype κ/IgG1) Fab fragment were investigated under conditions that have been reported to induce the AFS. Surprising differences in the ability of individual domains to form the AFS were observed, despite the similarities in their native structures. All Fab domains were able to adopt the AFS, but only for V_H (variable domain of the heavy chain) could a significant amount of tertiary structure be detected and different conditions were needed to induce the AFS. V_H, the least stable of the domains under physiological conditions, was the most stable in the AFS, yet all domains showed significant stability against thermal and chemical unfolding in their AFS. Formation of the AFS was found to generally proceed via the unfolded state, with similar rates for most of the domains. Taken together, our data reveal striking differences in the biophysical properties of the AFS of individual antibody domains that reflect the variation possible for domains of highly homologous native structures. Furthermore, they allow individual domain contributions to be dissected from specific oligomer effects in the AFS of the antibody Fab fragment.     

Localization of Proteinase(s) near the Cell Surface of Streptococcus lactis

Two criteria suggest that most of the proteinase of Streptococcus lactis is localized in the cell wall. (i) Intact cells possess proteinase activity when incubated with a high-molecular-weight substrate. (ii) Most of the cell-bound proteinase activity is released during spheroplast formation under conditions which result in the release of only 1% of the intracellular enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase. The solubilized cell wall, plasma membrane, and cytoplasm fractions contained 84, 0, and 16%, respectively, of the total proteinase activity with casein as substrate. The physiological role of a surface-bound proteinase in this organism is discussed.     

Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes.

Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.     

Antibodies to different peptides in osteopontin reveal complexities in the various secreted forms

We have generated synthetic peptides corresponding to various portions of human osteopontin (OPN) and have immunized rabbits and mice with these peptides to generate polyclonal and monoclonal antibodies specific to human OPN. We then generated six distinct sandwich enzyme-linked immunoabsorbent assay (ELISA) systems by using different pairs of polyclonal and monoclonal antibodies against human OPN. These systems allowed us to detect not only various isoforms and truncated forms of recombinant OPN, but also the glycosylated form of native urinary OPN. Most importantly, tumor-derived OPN was differentially detected by the six ELISA systems. The ELISA systems that we have developed will be useful for clarifying the functional roles for OPN in vivo in various physiologic and pathologic conditions.     

Immunochemical studies of estrogen receptors

Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from the cytosol of MCF-7 human breast cancer cells, with two different mouse myeloma lines, has provided 13 monoclonal hybridoma lines secreting antiestrophilin antibodies, each of which (with one possible exception) recognizes a different antigenic determinant in the human receptor molecule. Of this library of monoclonal antibodies, some react with estrophilin from all sources tested, some react with mammalian but not avian receptors, whereas one preparation appears specific for estrophilin from primate sources. By proteolytic digestion under controlled conditions with mercury-deactivated papain, chymotrypsin, and trypsin, respectively, it is possible to remove sequentially the determinants recognized by one, two or three of the monoclonal antibodies, leaving the epitopes for the six remaining antibodies investigated on the steroid-binding portion of the receptor. The proteolytic fragment containing the epitope most readily removed (by mercuripapain) also contains the DNA-binding domain of the activated receptor molecule. Immunocytochemical staining, using the peroxidase procedure with various monoclonal antibody preparations, of frozen sections of human breast cancer tissue, fixed in ethanol or in picric acid-formaldehyde reagent, shows clearly that the majority of the native receptor, which appears in the cytosol after tissue homogenization, is actually localized within the nuclear compartment in the intact cell. The immunocytochemical technique also permits the identification of mixed populations of receptor-containing and non-containing cells in human breast cancers.     

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.     

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site.

Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.