Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Purification and characterization of an abnormal factor IX (Christmas factor) molecule. Factor IX Chapel Hill

Human Factor IX (Christmas factor) was isolated from the plasma of a patient with mild hemophilia B. The patient's plasma contained 5% Factor IX clotting activity but 100% Factor IX antigenic activity as determined by immunological assays, which included inhibitor neutralization and a radioimmunoassay for Factor IX. This abnormal Factor IX is called Factor IX Chapel Hill (Factor IXCH). Both normal Factor IX and Factor IXCH have tyrosine as the NH2-terminal amino acid. The two proteins have a similar molecular weight, a similar amino acid analysis, the same number of gamma-carboxyglutamic acid residues (10 gamma-carboxyglutamic acid residues), and a similar carbohydrate content. Both exist as a single-chain glycoprotein in plasma. The major difference between normal Factor IX and Factor IXCH is that the latter exhibits delayed activation to Factor IXa in the presence of Factor XIa and Ca2+. Thus, Factor IXCH differs from other previously described abnormal Factor IX molecules.     

Purification and partial sequence of proteins involved in the cholic acid transport into rat liver hepatocytes

Two proteins, in previous work labeled by affinity markers derived from taurocholic acid, were purified and partially sequenced. Antibodies were raised against purified proteins, and cross-reactions were carefully checked. The influence of these antibodies upon taurocholic acid import into vesicles from rat liver plasma membranes was measured, and showed a distinct inhibition of transport in the case of the 54 kD protein.     

Prediction of solution structures of the Ca2+-bound gamma-carboxyglutamic acid domains of protein S and homolog growth arrest specific protein 6: use of the particle mesh Ewald method

The solution structures of the N-terminal domains of protein S, a plasma vitamin K-dependent glycoprotein, and its homolog growth arrest specific protein 6 (Gas6) were predicted by molecular dynamics computer simulations. The initial structures were based on the x-ray crystallographic structure of the corresponding region of bovine prothrombin fragment 1. The subsequent molecular dynamics trajectories were calculated using the second-generation AMBER force field. The long-range electrostatic forces were evaluated by the particle mesh Ewald method. The structures that stabilized over a 400-ps time interval were compared with the corresponding region of the simulated solution structure of bovine prothrombin fragment 1. Structural properties of the gamma-carboxyglutamic acid (Gla) domains obtained from simulations and calcium binding were found to be conserved for all three proteins. Analysis of the predicted solution structure of the Gla domain of Gas6 suggests that this domain should bind with negatively charged phospholipid surfaces analogous to bovine prothrombin fragment 1 and protein S.     

Sequence of the hexameric juvenile hormone-binding protein from the hemolymph of Locusta migratoria

The cDNA for the hexameric hemolymph juvenile hormone-binding protein (JHBP) from the migratory locust has been cloned and sequenced. Antiserum raised against purified JHBP was used to identify clones in an expression library. The 4.3-kilobase JHBP mRNA codes for 668 amino acids (74.4 kDa) and contains 2 kilobases of 3'-untranslated region. The derived amino acid sequence reveals that locust JHBP represents a new group within the hexamerin family of arthropod proteins. JHBP appears to be more closely related to arthropod hemocyanins, the believed ancestors of the family, than to the other known insect hexamerins. The mRNA shows a high (89%) bias to codons ending in G or C and the codons ending in A or T are clustered and concentrated toward the 5' end, suggesting a mosaic gene structure. The recombinant bacterially expressed protein bound [3H]JH III with the same affinity as the protein from hemolymph. A truncated version of JHBP lacking 53 amino acids from the N terminus did not bind JH III. Hybridization analysis of fat body JHBP mRNA in locusts that had been treated with precocene and a JH analog did not give clear evidence for regulation by JH.     

gamma-Carboxyglutamic acid in human urine

gamma-Carboxyglutamic acid, the amino acid responsible for the vitamin K dependent, Ca2+-binding structures of some of the blood coagulation proteins, has been identified in human urine. The amino acid wasisolated and its identity was proved by comparing it with synthetic gamma-carboxyglutamic acid by electrophoretic and chromatographic methods and by mass spectrometry. The isolated compound was also converted to glutamic acid by heat decarboxylation, a reaction consistent with its anticipated structure. A method for the quantitation of free gamma-carboxyglutamic acid in human urine was developed. The method consisted of an anion-exchange concentration step followed by automatic amino acid analysis using a pH 2.0 lithium citrate buffer. In three non-fasting adult males the urinary excretion of gamma-carboxyglutamic acid ranged between 27 and 42 mumol/24 h and in six non-fasting adult females it ranged between 19 and 32 mumol/24 h. One fasting adult male excreted 36 mumol/24 h.     

A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a beta-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase. p27 exhibits isomerase activity with a kcat/Km of 0.18 microM-1 s-1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.     

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi

The amino acid gamma-carboxyglutamic acid (Gla) is found in four blood-clotting proteins, in a bone protein, in kidney protein, and in the protein present in various ectopic calcifications. This paper reports the presence of Gla in the EDTA-soluble, nondialyzable proteins of calcium-containing renal calculi including calcium oxalate, hydroxyapatite, and mixed stores of apatite and struvite (MgNH4PO4). Calculi composed of pure struvite and those composed of only uric acid or cystine do not contain Gla. From calcium oxalate and hydroxyapatite stontes, a protein of about 17,000 daltons was obtained which contained about 40 residues of Gla per 1,000 amino acids. The amino acid composition of this protein had no apparent relationship to the Gla-containing bone protein or to the similarly-sized F1 fragment of prothrombin which contains about 64 residues of Gla per 1,000 amino acid residues. The Gla-rich protein in calcium-containing renal stones thus may be a different Gla-containing protein. These data as well as other studies demonstrating the presence of Gla in pathologically calcified tissues not normally containing Gla suggest that the Gla-containing proteins may be of considerable pathophysiological significance.     

Purification and properties of two blue biliproteins from the larval hemolymph and integument of Rhodinia fugax (Lepidoptera: Saturniidae)

Blue biliproteins (BPs) are found in the hemolymph and integument of the fifth instar larvae of the saturniid silkworm, Rhodinia fugax. An efficient method of isolating BPs from the hemolymph, epidermis and cuticle using hydrophobic interaction chromatography and ion-exchange chromatography was devised. The BPs from the hemolymph, epidermis and cuticle have molecular weights of approximately 24,000, 48,000 and 23,000 Da by gel-filtration, respectively. Using matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS), the respective molecular masses were determined to be 22,641, 22,908 and 22,737 Da. Based on these results, BP molecules from the hemolymph and cuticle are assumed to be monomers, whereas the epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from the hemolymph and cuticle (BP-I) are very similar, but the BP from the epidermis (BP-II) is quite different. The N-terminal amino acid sequences of these BPs share approximately 50% identity with the biliproteins from other lepidopteran insects. The blue color of BP is due to the presence of bile pigments, which are non-covalently bound to the apoprotein. The absorbance spectrum of BP-I from the hemolymph revealed maxima at 280 and 669 nm, while that of BP-II showed maxima at 280, 385 and 663 nm. The pigment dimethyl esters were extracted from BP-I and BP-II with acidic methanol and dichloromethane. The results of these analyses suggest that the blue pigments of BP-I and BP-II are different; BP-I contains a phorcabilin-like pigment while BP-II contains biliverdin IX gamma. In an immunoblot analysis, anti-BP-I antibodies, produced against hemolymph BP-I, reacted with immunoreactive proteins in the hemolymph and cuticle of R. fugax. These anti-BP-I antibodies did not react with BP-II and only cross-reacted weakly with Samia cynthia ricini biliverdin-binding protein (BBP)-II.     

Urinary stone proteins: an update

The discovery of an organic component in kidney stones dates back to 1684. More than 150 years elapsed before the incrustation of this organic component, which is now called the matrix, was proposed as the mechanism of stone formation. The composition of the matrix remained largely unknown until the development of electron microscopy and the advances in biochemistry combined in the 1950's to usher in the modern era of renal stone matrix investigation. Composed mainly of selectively incorporated proteins generally characterized by high glutamic and aspartic acid content and the frequent occurrence of gamma-carboxyglutamic acid, the matrix displays a variable and complex composition and shares a few proteins in many stones. The embryonic stone may first appear in the renal tubules where it can acquire the blood and cell membrane proteins recently found by analysis of stone protein extracts. The combination of supersaturation, an appropriate environment, the availability of calcium binding proteins which may be abnormal, and the incorporation of proteins extracted from leukocytes and cell wall membranes may induce stone formation.     

Subunit composition of pro-phenol oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1

Phenol oxidase (PO) is known to play an important role in defense mechanisms in insect immunity. It is present as a zymogen in insect hemolymph, and can be activated by a specific proteolytic reaction that is stimulated by microbial cell wall components. The pro-phenol oxidase (pro-PO) purified from the larval hemolymph of Manduca sexta contains two polypeptides in equal amounts as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cDNA for one of the polypeptides, now designated proPO-p2, has been isolated (Hall et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 7764-7768). We purified pro-PO from plasma of M. sexta and characterized its subunit composition. A cDNA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA library. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bombyx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immunofluorescence labelling and in situ hybridization showed that the pro-PO is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multimeric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a detergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of distinct mobility (fast and slow forms). Both forms contain proPO-p1 and proPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indicate that there are two pro-PO genes in M. sexta, which are coordinately expressed in oenocytoids, and whose products form predominantly heterodimers in plasma.     

Urinary protein excretion in red jungle fowl (Gallus gallus)

Urinary protein excretion by red jungle fowl (Gallus gallus) was examined by measuring total protein concentrations in the ureteral urine and by comparing the proteins in the urine with plasma proteins. Protein concentration in the ureteral urine did not differ between males and females, and averaged 2.01 mg/ml. Gel electrophoresis showed many plasma proteins (30-149 kD) also present in the urine. Serum albumin is the most abundant protein, comprising approximately 50% of the total protein concentration in the plasma and 60% of the total protein concentration in the urine. Urinary protein, and particularly serum albumin, may be important in packaging uric acid in spheres, which facilitates excretion of uric acid without formation of large crystals that could block renal tubules.     

Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae

The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus gene have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich VHv1.4 protein. Full-length and truncated VHv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the VHv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The VHv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The VHv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the VHv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the VHv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the VHv1.4 protein was internalized, probably by endocytosis. Specific binding of the VHv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

Purification and partial characterization of vitellin from the black-legged tick, Ixodes scapularis

Vitellin from the black-legged tick, Ixodes scapularis, was purified from eggs using gel filtration and ion exchange chromatography. The purified protein had a native molecular mass of 480 kDa. Under reducing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS-PAGE), vitellin was composed of seven polypeptides each at 154, 135, 87, 78, 67, 64 and 35 kDa. The isoelectric point was pH 6.9 and absorption maxima for the yolk protein were 280 and 400 nm. As in other ticks, vitellin from I. scapularis is also a hemoglycolipoprotein. Carbohydrates detected in vitellin were predominantly mannose with a small amount of N-acetylglucosamine. Lipids detected by thin layer chromatography (TLC) were triglycerides, free fatty acids, and cholesterol. Phospholipids associated with vitellin were phosphatidylethanolamine and phosphatidylcholine. Polyclonal serum produced in rabbits recognized vitellin from the eggs and ovaries, and vitellogenin from the hemolymph and fat body in reproductive females. This is the first report on the characterization of yolk proteins from a prostriate tick.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.     

Purification and partial characterization of 76 kDa transglutaminase in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss

Transglutaminase (TGase), responsible for crosslinking between proteins, is known to be localized exclusively in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss, and probably participates in the post-fertilization chorion hardening. We purified the TGase from unfertilized egg chorions by sequential chromatography using SP-Sepharose, Q-Sepharose, and TSK-gel G3000SWXL columns. The purified enzyme was a monomeric protein having the molecular mass of 76 kDa. It promoted incorporation of monodansyl-cadaverine into chorion protein and catalyzed the polymerization of chorion subunit proteins. The effect of various reagents suggested that the chorion TGase is a Ca2+-dependent SH-enzyme similar to the well-characterized TGases of various animals. The highest activity was observed at pH 6.0. The amines examined in the present study inhibited the TGase activity of the purified enzyme. However, they did not necessarily cause effective inhibition of its activity. These properties of the chorion TGase were essentially consistent with our previous observations on polymerization of chorion proteins, resulting in chorion hardening. We compared the amino acid composition of the purified TGase with those of the previously characterized TGases of fishes, such as chum salmon and red sea bream. The results suggest that the chorion 76 kDa TGase is not homologous with those liver TGases in terms of amino acid composition.     

Mass-spectrometric identification and sequence location of the ten residues of the new amino acid (gamma-Carboxyglutamic acid) in the N-terminal region of prothrombin

The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.     

Purification and characterization of a human bradykinin binding protein from inflammatory cells

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.     

Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma

Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.