NH2-terminal Sequence Analysis Research Articles

Plasma Procofactor Activation by Factor Xla

The procofactors FV and FVIII are activated by thrombin, FXa and plasmin. During contact pathway-initiated thrombin generation, FXIa activates FIX thus feeding into the coagulation cascade; however, the procofactors FVIII and FV must be activated to achieve a robust level of thrombin generation. We tested the hypothesis that FXIa can activate FV and FVIII. FV (1uM) was subjected to FXIa (100nM) proteolysis. During the reaction the relative activity and integrity were measured at selected time points using a one stage PT clotting assay and SDS-PAGE. Over the 60 minute time course, FV showed a transient 50% activation followed by a reduction in activity to 20%. SDS-PAGE analyses showed that proteolysis of FV by FXIa initially generated fragments with mobilities similar to those produced by α-thrombin. This activation and inactivation pattern suggested that FXIa makes the required activation cleavages at R709, R1018 and R1545 coincidently with inactivating proteolyses. FV was activated with α-thrombin and the reaction quenched by the addition of hirudin prior to FXIa proteolysis. FVa's cofactor activity was reduced by 50% after 30 min and 80% after 60 min. Analysis of the FXIa cleavage process by SDS-PAGE under reducing conditions showed no intact heavy chain and significant proteolysis of the light chain after 30 minutes. In addition to the Mr = 105000 (HC) and Mr = 75000 (LC), six new products were identified by SDS-PAGE under reducing conditions: Mr = 54000, 50000, 48000, 30000, 22000 and 20000. NH2-terminal sequence analysis indicated a single cleavage in the light chain at R1765 yielding the products Mr = 50000, 48000: (1766à2196) and the Mr = 30000 (1546à1764), also seen with plasmin and FXa. A cleavage in the heavy chain represented by the Mr = 22000, 20000 (R511à709) fragments is also observed. Sequence analysis determined that the Mr = 54000 fragment represented the NH2-terminus of the heavy chain. Western analysis using a heavy chain antibody showed a transient band Mr = 75000 lying under the light chain that is consistent with an initial cleavage R510. A subsequent cleavage, which is coincident with a decrease in the cofactor's activity, results in a Mr = 30000 product which is consistent with a cleavage somewhere COOH-terminal to R306(R316?). Activity analyses suggest that the initial cleavage in the heavy chain at R510 leads to a FVaXla 1 molecule with similar activity (50%) to that seen in FVa cleaved by APC in the absence of phospholipid. The FVaXla 1 was treated with APC resulting in complete inactivation of the cofactor. We have also observed an analogous FXIa cleavage pattern in FVIII; however sequencing analysis has not yet been attempted. These data suggest that factor XIa may play a role in procofactor activation and inactivation of FV and FVIII in the context of contact pathway-initiated blood coagulation. [Display omitted]

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-α2b) is the most widely used member of IFNα family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-α2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-α2b has been achieved. SDS–PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-α2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex™ 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1 l of the supernatant and its identity to hIFN-α2b was confirmed by NH 2-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 × 10 9 IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-α2b for both research and industrial purpose.

Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS–PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE–Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH 2-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.

우유로부터 Osteopontin의 분리.정제 및 특성에 관한 연구

본 연구는 우유로부터 OPN을 분리 정제하여 그 특성을 규명하기 위해서 수행되었다. 먼저 ion-exchange와 hydrophobic chromatography를 이용하여 우유로부터 OPN을 분리 정제하였다. OPN의 분자량은 SDS-전기영동 상에서 약 60,000dalton이었고, NH2-terminal amino acid sequence를 확인한 결과 Leu-Pro-Val-Lys-Pro- Thr-Ser 순이었다. OPN을 35주령된 SCWL를 이용하여 산란계를 면역 시키고, 형성된 anti- OPN IgY 항체를 분리․정제한 후 ELISA test로 항체가를 측정하였다. 또한 RID test을 이용하여 OPN 함량에 따른 정량곡선을 작성하고, 이 곡선에 의해 우유 중 OPN 함량을 정량하였다. 그 결과 원유, 탈지유, 시유에서 각각39.78, 31.74, 37.48<TEX>${\mu}g$</TEX>/<TEX>$m\ell$</TEX>을 함유하고 있는 것으로 나타났다. 또한 OPN의 Ca 가용화 능력을 검정한 결과 OPN이 CPP와 poly-glutamic acid 보다 더 우수한 것으로 나타났다. The purpose of this study is to observe purification and properties of osteopontin(OPN) from bovine milk. The purification of osteopontin from bovine milk was performed by using ion-exchange and hydrophobic chromatography. SDS-PAGE analysis revealed that the protein migrated at Mw. 60,000. NH2-terminal sequence analysis of the first seven amio acids revealed the protein to be identical to that previously reported for bovine OPN. 35-wk-old chickens, including 3 Single Comb White Leghorn (SCWL), were used to produce egg yolk antibody(IgY) against OPNas a antigen. However, the anti-OPN antibody activities determined by ELISA. Immunological assy of OPN in milk was performed using radial immunodiffusion test based on the standard curve of pure OPN. The radial precipitation lines of four different milk samples indicated that the concentrations of OPN in the milk samples were within the range of 31.7 to 39.7<TEX>${\mu}g$</TEX>/ml. On inhibition with OPN on precipitation of calcium phosphate, OPN was slightly higher than casein phosphopeptide(CPP) and poly-glutamic acid.

Identification of interleukin-8 converting enzyme as cathepsin L

IL-8 is produced by various cells, and the NH 2-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH 2-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH 2-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg 5 and Ser 6, which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

Purification and characterization of human soluble CD14 expressed in Pichia pastoris

CD14 is a protein that mediates lipopolysaccharide (LPS)-induced biological responses such as activation of a transcriptional factor, nuclear factor (NF)-κB. It exists as a soluble form (sCD14) in serum and mediates LPS responses of epithelial and endothelial cells as well as a membrane-bound form (mCD14) on monocytes and macrophages. To obtain sCD14 in large quantity for its structural and functional characterization, we expressed the full-length form of human recombinant sCD14 (rsCD14) in a methylotrophic yeast, Pichia pastoris. The recombinant protein was expressed as a major protein in the culture supernatant and purified by ammonium sulfate precipitation, followed by three steps of ion exchange chromatographies. Finally, 1.6 mg of the protein was obtained in high purity from 2 L of the supernatant and its identity to sCD14 was confirmed by NH 2-terminal amino acid sequence analysis. The purified protein was found to have N-linked sugars by an analysis of enzymatic deglycosylation. A native PAGE analysis revealed that the protein was able to form complexes with LPS. In addition, the rsCD14 protein could mediate the LPS-mediated activation of NF-κB in human embryonic kidney 293 cells transfected with Toll-like receptor 4 and MD-2, indicating that the purified protein is biologically active. Thus, the rsCD14 protein expressed in P. pastoris and highly purified in a large amount is useful for its structural and functional studies.

Production of two types of phytase from Aspergillus oryzae during industrial koji making

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an important role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH2-terminal sequence of Phy-1, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy-I, 120 kDa; ACP-II, 58 kDa). From the NH2-terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p-nitro phenyl phosphate, but also glucose 6-phosphate and glycerol 1-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.

Purification and some properties of cholesterol oxidase stable in detergents from γ-proteobacterium Y-134

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the γ-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30°C for 12 d. It was purified from cell-free extract prepared by mixing the cells with 0.4% Triton X-100. The absorption spectrum of the purified enzyme exhibited maxima at 274 and 410 nm, and a shoulder at 330 nm. The molecular mass was 115 kDa with two identical subunits of 58 kDa. The enzyme oxidized cholest-5-en-3β-ol (cholesterol) and 5α-cholestan-3β-ol (dihydrocholesterol) at a high reaction rate, and the K m value for cholesterol was 65 μM. The stability of the enzyme was higher than other CHOs in nonionic detergents with high values of hydrophilelipophile balance (HLB) such as Triton X-450 and sodium cholate. NH 2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.

Two subtilisin-like proteases from soybean.

Two subtilisin-like proteases (SLP) were identified in soybean (Glycine max [L.] Merr.). The first, SLP-1, was localized in seed coats early in seed development, but became undetectable with anti-SLP-1 antibodies as seed fill progressed. A partial purification of SLP-1 was achieved using a two step chromatographic procedure. NH2-terminal sequence analysis of the partially purified enzyme permitted primers to be designed that were used to amplify cDNA encoding SLP-1. A genomic clone encoding SLP-1 was also obtained. Characterization of the cDNA and partially purified SLP-1 revealed the initial translation product was an 82 694 MW precursor. After removal of a signal peptide, the mature protein was formed by removal of an NH2-terminal propeptide. A COOH-terminal peptide also appeared to be removed from some of the protease molecules. DNA blot analysis suggested that at least one additional SLP gene was present in soybean. The second gene, SLP-2, was subsequently cloned and characterized. Although the coding regions for SLP-1 and SLP-2 were homologous, their promoters were quite divergent. RT-PCR revealed that SLP-2 message was found in the mature plant and in cotyledons of germinating seeds. Although SLP-2 mRNA could be identified in developing seeds, the message was at least an order of magnitude less abundant than that for SLP-1, and it was mis-spliced such that a chain termination event would preclude obtaining a product. As with SLPs from other organisms, the functions of the soybean proteases are unknown. However, SLP-1 is one of only a few proteins from soybean seed coats that have been described.

Identification of C3a and N-truncated C3a as vascular permeability-enhancing factors from the exudate of chronic phase of carrageenan-induced inflammation in rats.

Two basic proteins enhancing vascular permeability have been purified from the exudate of the chronic phase of carrageenan-induced inflammation in rats. One major and one minor peak on reversed-phase HPLC showed molecular masses of 9.3 kDa and 7.6 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. NH2-terminal amino acid sequencing analysis of the purified proteins revealed that the major peak is identical to C3a, while the main sequence of the minor peak is identical to NH2-terminal 11 amino acids truncated C3a. In addition, plasmin was able to cleave C3a into the N-truncated C3a. Intradermal injection of both purified C3a and N-truncated C3a into rat skin enhanced vascular permeability, and the increased permeability was suppressed by the pretreatment with cyproheptadine. Our results suggest that the purified C3a and N-truncated C3a have the characteristics of anaphylatoxins and may contribute to exudation in the chronic phase of carrageenan-induced inflammation in rats.

Molecular identification of IgE-dependent histamine-releasing factor as a B cell growth factor.

The culture supernatants of LK1 cells, murine erythroleukemia cells, showed B cell-stimulating activity. Purification and NH(2)-terminal sequence analysis revealed that one of the candidates was murine IgE-dependent histamine-releasing factor (IgE-HRF), which is known to induce histamine from basophils. Recombinant IgE-HRF (rHRF) obtained from Escherichia coli- or 293-transformed embryonal kidney cells was tested for B cell-stimulating activity. Both rHRFs stimulated B cell proliferation in a dose-dependent manner. However, boiling or anti-HRF Ab abolished the B cell stimulatory effects of rHRF. Recombinant HRF showed strong synergistic effects with IL-2, IL-4, and IL-5 for B cell activation, with maximal activity in the presence of anti-CD40 AB: Recombinant HRF increased MHC class II expression of B cells. It also increased Ig production from B cells. Treatment with polymyxin B, a neutralizing peptide antibiotic of LPS, did not reduce the activity of rHRF. In addition, FACS analysis using PE-conjugated rHRF showed that HRF bound to B cells. Recombinant HRF up-regulated the expression of IL-1 and IL-6 in B cells. In vivo administration of rHRF or the cDNA for rHRF increased total and Ag-specific Ig synthesis. Taken together, these results indicate that HRF stimulates B cell activation and function.

The Conformation of Calreticulin Is Influenced by the Endoplasmic Reticulum Luminal Environment

The Conformation of Calreticulin Is Influenced by the Endoplasmic Reticulum Luminal Environment

Matrix Metalloproteinases Cleave Tissue Factor Pathway Inhibitor: EFFECTS ON COAGULATION

Matrix Metalloproteinases Cleave Tissue Factor Pathway Inhibitor: EFFECTS ON COAGULATION

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS–PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH 2-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.

Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors

A novel serine protease (PHBSP) was purified from human plasma by two chromatographic steps with a final yield of 1.6 mg/l plasma. The protease consists of two disulfide-bridged chains of about 50 and 30 kDa with the light chain containing the active site of the enzyme. NH 2-terminal sequence analysis revealed identity to the deduced amino acid sequence of HGFA-like mRNA. The activity of PHBSP is strongly dependent on Ca 2+ ions and is efficiently inhibited by α 2-antiplasmin and aprotinin. Possible functions of PHBSP in the hemostatic system are discussed.

Occurrence of heavy chain of 7S IgM half-molecule whose NH 2-terminal sequence is identical with that of κ light chain sequence in patients with Waldenstrom macroglobulinemia

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique μ heavy chain and κ light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH 2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1–20 were identical to those of the NH 2-terminal region of κ light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique μ chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known μ chain to residue 19 in the known CH 1 domain and a sequence spanning from residues 67–82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a κ light chain and a unique μ heavy chain of 35 kDa polypeptide in which the NH 2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of κ light chain in the NH 2-terminal region.

Photorhabdus luminescens W-14 Insecticidal Activity Consists of at Least Two Similar but Distinct Proteins: PURIFICATION AND CHARACTERIZATION OF TOXIN A AND TOXIN B

Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.

Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death.

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the alpha-ketoglutarate dehydrogenase complex (alphaKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of alphaKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of alphaKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.