Sequence homologies between tonin, nerve growth factor gamma-subunit, epidermal growth factor-binding protein and serine proteases.
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Amino Acid Sequence↗
Biomedical subjects
C Lazure
Publications and source records attributed to C Lazure.
Sequence diversity within a subgroup of mouse immunoglobulin kappa chains controlled by the IgK-Ef2 locus.
We previously showed that a chromosome 6 locus, IgK-Ef2, controls a pair of prominent bands in normal mouse light-chain isoelectric focusing profiles. Screening of myeloma light chains derived from BALB/c mice (an IgK-EF2 alpha strain) led to the identification of seven light chains cofocusing with the polymorphic bands controlled by IgK-Ef2. Complete sequencing of the variable (V) regions of four of the light chains indicates that they are all members of the same subgroup (Vk-1A) and they differ from one another by 1--3 substitutions. One of the protein differs from the prototype V-region sequence only in the deletion of a single residue at position 95 immediately preceding of J region. The other two differ from the protype V region by 3 (two framework [fr], one complementarity-determined [cdr]) and one (fr) residues, respectively. Complete V-region sequences of two closely related light chains derived from NZB mice (an IgK-Ef2b strain) indicate the NZB proteins are derived from a distinct Vk gene (Vk-1B), differing by four substitutions from the Vk-1A sequence. The results suggest that the IgK-Ef2 polymorphism may be a result of, at least in part, the loss of the gene(s) coding for the Vk-1A subgroups in IgK-Ef2b strains of mice. The nature of the sequence diversity found in the Vk-1A subgroup indicates that either it is coded by a repeated series of virtually identical genes or that somatic mutation of a single Vk-1A gene may give rise to substitutions in framework as well as cdr regions.
Amino Acid Sequence↗
A major group of mouse kappa chains controlled by the chromosome 6 locus, IgK-Ef2.
We previously demonstrated that loci closely linked to the Ly-3 locus control the expression of distinct sets of light chains in normal mouse serum immunoglobulin. One of these loci, IgK-Ef2, was shown to control two major bands in normal light chain isoelectric focusing (IF) profiles. Strains possessing the marker bands were designated IgK-Ef2a. Screening of myeloma proteins from the strains BALB/c (IgK-Ef2a) and NZB (IgK-Ef2b) led to the identification of eight proteins in the BALB/c collection having light chains that cofocus precisely with the polymorphic IF bands observed in normal serum light chains. Partial sequence analysis of 3 of the light chains has shown that they are all identical in the first 30 positions, which indicates that they constitute a single variable region of the kappa light chain (VK) group (VK1). The frequency of occurrence of the group within the BALB/c myeloma collections (8 out of 277) suggests that the number of such groups may be closer to 50 than to 100. The finding supports an interpretation of the genetic polymorphism as being in part a result of the absence of genes related to VK1 in IgK-Ef2b strains of mice.
Amino Acid Sequence↗
Isolation from human seminal plasma of an abundant 16-kDa protein originating from the prostate, its identification with a 94-residue peptide originally described as beta-inhibin.
In addition to other known markers of the human prostate, it was shown that the prostatic fraction of the split ejaculate was rich in a 16-kDa protein with properties not described previously. This protein was purified from human seminal plasma using ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and gel filtration on Sephadex G-100. The purified protein showed a single prominent spot on two-dimensional gel electrophoresis. The sequence of the first 40 amino acids that could be positively identified was identical to that of a prostatic secretory protein of 94 amino acids (PSP94) previously designated as beta-inhibin. Antibodies produced in rabbits against the purified protein were used to develop a radioimmunoassay. These antibodies appeared to recognize only the NH2-terminal portion of the native molecule since they did not react with a synthetic peptide composed of the 28 C-terminal residues. The radioimmunoassay showed that the concentration of the protein was 1320 +/- 183 micrograms/ml in the seminal plasma of adult fertile men and 1134 +/- 136 micrograms/ml in vasectomized patients. In hypertrophic and adenocarcinomatous prostates, the concentrations were 326 +/- 156 and 104 +/- 23 micrograms/ml, respectively, while values were lower than 0.060 micrograms/ml in the testis, epididymis, vas deferens and liver. The blood plasma concentration was 0.019 +/- microgram/ml in 23 asymptomatic men 45 to 65 years old and 0.115 +/- 0.036 microgram/ml in eight patients with prostate cancer.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Application of the multiple antigenic peptides (MAP) strategy to the production of prohormone convertases antibodies: synthesis, characterization and use of 8-branched immunogenic peptides.
Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediately following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84-99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, 1H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventional method of peptide-carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the case of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.
Amino Acid Sequence↗
NH2-terminal fragment of rat pro-atrial natriuretic factor in the circulation: identification, radioimmunoassay and half-life.
A radioimmunoassay was developed to measure the NH2-terminal counterpart of rat pro-atrial natriuretic factor (pro-ANF) in plasma. Synthetic rat ANF (Asp 11-Ala 37) coupled to bovine serum albumin was used to immunize New Zealand rabbits. The antiserum demonstrated good immunoreactivity towards rat ANF (Asn 1-Arg 98), (Asn 1-Tyr 126), (Asp 11-Ala 37) and even human ANF (Asn 1-Ser 30). The standard curve had an ED80 of 9.5 +/- 2.5 and ED50 of 44.0 +/- 10.5 fmol/tube. Immunoreactive ANF NH2-terminal peptide was measured directly in rat plasma without prior extraction. In fact, extraction of ANF NH2-terminal from plasma by C18 silica gel chromatography revealed inconsistent recovery and a lack of parallelism. Morphine (0.75 mg/100 g), chosen to elicit increased ANF (Ser 99-Tyr 126) secretion, elevated its plasma concentration from 54.1 +/- 3.2 to 190.8 +/- 55.8 fmol/ml after 20 min. At the same time, the immunoreactive NH2-terminal fragment rose from 378 +/- 16 to 1181 +/- 201 fmol/ml. The identity of this immunoreactive material was verified following affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC) of plasma from morphine-treated rats. Molecular sieving and amino acid sequencing demonstrated that it appears to be consistent with or identical to rat ANF (Asn 1-Arg 98). The disappearance rate of ANF (Asn 1-Arg 98) was studied by injecting radioactive material into anesthetized rats. The exponential decay was analyzed by a two-compartment model in which the fast and slow components had a half-life of 2.5 +/- 0.3 and 54.8 +/- 3.9 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(1-76).
A major peptide related to the NH2-terminal fragment (position 1 to 76) of mammalian chromogranin A was isolated from ostrich adenohypophyses following acid-acetone extraction. The complete amino acid sequence of the homogenous peptide was deduced following automatic Edman degradation of the native peptide as well as of CNBr-, tryptic- and Lysobacter-derived peptides. The 76 amino acid sequence is strikingly homologous to bovine (80.3% sequence identity), porcine (79.0%), human (79.0%) and rat (72.4%) corresponding sequences, but much less so to human chromogranin B (22.4%). As this peptide is followed in bovine, porcine and human structure by a pair of basic residues (Lys-Lys), it could conceivably be produced during maturation in secretory granules. Finally, its structure appears to contain two potential amphipathic helices joined by the single disulfide bridge present in all chromogranin A and B molecules.
Amino Acid Sequence↗
The production of the ostrich NH2-terminal POMC fragment requires cleavage at a unique signal peptidase site.
The NH2-terminal fragment of ostrich proopiomelanocortin was isolated and purified following acid/acetone extraction. The amino acid sequence was deduced by automatic Edman degradation of the native as well as CNBr-, tryptic-, and S. aureus protease-derived peptides. Primary structure analysis reveals its close resemblance to other known sequences, especially to amphibian POMC. The usual Trp/Gln-Cys NH2-terminal sequence found in all other homologous sequences, is replaced here by an His-Gly-Pro-Cys sequence. In addition, the gamma-MSH sequence, contrary to salmon POMC, is present and contains three substitutions, namely a Ser, an Asn, and a Lys residue substituting the normally occurring mammalian Gly, Asp, and Arg residue, respectively. Finally, the molecular weight of this fragment as deduced from ion-spray mass spectrometry and sedimentation equilibrium centrifugation is in close agreement with the proposed structure.
Amino Acid Sequence↗
Structure and genetic control of V kappa-1 light chains.
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Amino Acid Sequence↗
Biotinylation of an enkephalin-containing heptapeptide via various spacer arms. Synthesis, comparative binding studies toward avidin, and application as substrates in enzymatic reactions.
The preparation of an enkephalin-containing heptapeptide of the sequence Tyr-Gly-Gly-Phe-Leu-Arg-Arg-OH with a biotinyl moiety linked to the carboxy terminus is described. A series of biotinylated derivatives, each containing a different linker (LC) moiety between the biotin function and the carboxyterminal Arg residue, were synthesized by solution-phase chemistry following the coupling of the side chain protected peptide with previously prepared appropriate biotinylamine derivative. Both linear and flexible spacer arms of variable chain lengths [LC = (CH2)x, x = 2, 4, or 6] as well as semirigid cyclohexyl spacers (racemic 1,2-cyclohexane, cis or trans) were incorporated. The relative binding aptitudes of these molecules toward the glycoprotein, avidin, either in immobilized form or in solution were compared using both 125I-labeled and unlabeled peptide derivatives and were found to be in the following order, trans > or = 6C > 4C > 2C > cis. The potential application of these materials as substrates for enzymatic analysis is illustrated for one of the derivatives, namely the LC-2C analogue.
Amino Acid Sequence↗
Amino acid sequence of rat submaxillary tonin reveals similarities to serine proteases.
Tonin, an esteroprotease isolated from rat submaxillary gland, is a serine protease with trypsin- and chymotrypsin-like activity. The substrate specificity of tonin shows that it differs from kallikreins and is definitely not a renin-like enzyme or an angiotensin-converting enzyme. Tonin can produce directly the vasoactive peptide angiotensin II, from angiotensin I, angiotensinogen and the synthetic tetradecapeptide substrate of renin by cleavage of a Phe-His bond. It has also been found to cleave some Phe and Arg bonds in various substrates such as beta-lipotropin (beta-LPH), adrenocorticotropin (ACTH), pro-opiomelanocortin (POMC) and substance P. Here we describe the complete amino acid sequence of rat submaxillary gland, tonin. Comparison of the sequence of 219 amino acids with other serine proteases, particularly kallikreins, gamma-subunit of nerve growth factor (NGF) and the recently described gamma-renin, reveals extensive similarities. More interestingly, it also reveals the substitution of an Asp residue always found in the serine protease active site triad (Asp, His, Ser) by a Leu residue. This unusual substitution does not seem to affect the proteolytic activity of the enzyme.
Amino Acid Sequence↗
Peptidyl substrates containing unnatural amino acid at the P'1 position are potent inhibitors of prohormone convertases.
In order to study further the importance of the P'1 residue upon the activity of human PC1 and human furin, two important members of subtilisin/kexin family of enzymes, we have prepared by solid-phase Fmoc or recently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs. The structures of these analogs are based upon the core sequence of pro-mPC1(83-93) namely, D-Tyr-Lys-Glu-Arg-Ser-Lys-Arg-Xaa-Val-Gln-Lys-Asp, where D-Tyr replaces the native L-Tyr residue and Xaa, representing the P'1 position, corresponds to L-Ser or to nonproteinacous amino acids such as Tle, Sarc, MLeu, Aib, D-Tic or L-Tic. Two more analogs with L-Tic at P'1 position but with one amino acid less, namely P5 Glu or P'3 Gln, and one with a Cit residue in place of Arg at P1 site of the dodecapeptide were also obtained. These peptides were all fully characterized by a combination of MS, 1H-NMR and amino acid analysis. In contrast to the Ser analog, which is an excellent substrate for both hPC1 and hfurin, these analogs displayed moderate to strong inhibition of both hPC1 and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPC1, with an inhibition constant (Ki) ranging from 0.8 to 10 microM and from 1.0 to 170 microM, respectively. Incorporation of L-Tic yielded an analog with a two to four-fold increased inhibition of either enzymes when compared to its D-Tic counterpart, the effect being more pronounced for hPC1 than for hfurin. Comparison of these data with those for the corresponding N-terminal Fmoc protected peptides revealed that the highly hydrophobic N-terminal Fmoc function occupying the P8 position can contribute positively or negatively towards proteinase inhibition depending on the nature of the unnatural amino acid at P'1 and the enzyme used. Finally, none of the analogs was significantly cleaved by either enzyme. FTIR data on these analogs revealed some important structural differences between the substrate and inhibitor analogs, as there appears to be a conformational shift from a more beta-sheet-like structure for the substrates to a more alpha-helical-like structure for the inhibitors.
Amino Acid Sequence↗
High concentrations of the macrophage migration inhibitory factor in human seminal plasma and prostatic tissues.
During purification procedures to isolate kallikrein hK2 from human seminal plasma, kallikrein hK2 was found to be associated with another protein after several chromatographic steps. This study was conducted to identify the hK2 companion protein and characterize its properties and distribution. The protein was identified as macrophage migration inhibitory factor (MIF) by its NH2-terminal amino acid sequence. It had an enzymatic activity identical to that of recombinant MIF. Its concentration varied between 1 and 10 micrograms/mL in various seminal plasma. By immunohistochemical analysis, MIF was found to be localized mainly in the epithelial cells of normal and cancerous prostates. Since MIF is a well-known proinflammatory mediator, these results suggest that it may have important functions in both human reproduction and prostatic physiology.
Amino Acid Sequence↗
Urokinase-type plasminogen activator: a paracrine factor regulating the bioavailability of IGFs in PA-III cell-induced osteoblastic metastases.
The transplantation of PA-III rat prostate cancer cells onto rat skeleton produces osteoblastic metastases. Therefore w e studied the paracrine interactions between the PA-III cells and osteoblast-derived osteosarcoma cells (UMR 106 cells). A serine protease secreted by PA-III cells hydrolyzed IGF-binding protein-1 and IGF-binding protein-2 (IGFBP-1 and IGFBP-2) detected in the cell culture media (CM) of OMR 106 cells by western ligand blotting. The serine protease of PA-III cell CM was purified using a benzamidine affinity column. This protease was a protein of 45-50 kDa on polyacrylamide gel electrophoresis under non-reducing conditions but generated two protein bands under reducing conditions; a) one of 33-35 kDa possessing protease activity and b) another of 20-25 kDa which was proteinolytically inactive. Sequence analysis identified the amino acid sequence of the a-chain (20-25 kDa band) and of the b-chain (33-35 kDa band) of rat urokinase-type plasminogen activator molecule. Urokinase purified from PA-III cell CM hydrolyzed IGFBPs of UMR 106 cells and stimulated the proliferation of UMR 106 cells in serum-free cultures. Its protease activity was abolished by benzamidine and aprotinin. Its mitogenic activity for osteoblasts was inhibited by anti-IGF-I monoclonal antibody. Northern blot analysis documented the expression of the urokinase-type plasminogen activator gene in the mRNA extracted from PA-III cells. Urokinase expression was inhibited by dexamethasone. Therefore, we conclude that urokinase-type plasminogen activator stimulates osteoblasts via an IGF-I dependent mechanism. Hydrolysis of the IGFBOPs at the sites of PA-III cell-induced bone tumors account for an increased bioavailability of IGFs. This may facilitate the development and the growth of PA-III cell-induced bone tumor and can also mediate the subsequent local osteoblastic reaction.
Animals↗