RESUMEN

SUMMARY: A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Asunto(s)

Factor VIII/química , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cricetinae , Factor VIII/aislamiento & purificación , Glicoproteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismo

RESUMEN

The mammalian protease plasminogen can be activated by bacterial activators, the three-domain (alpha, beta, gamma) streptokinases and the one-domain (alpha) staphylokinases. These activators act as plasmin(ogen) cofactors, and the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have investigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus uberis (Sk(U)) with specificity toward bovine plasminogen. The interaction between Sk(U) and plasminogen occurred in two steps: (1) rapid association of the proteins and (2) slow transition to the active complex Sk(U)-PgA. The complex Sk(U)-PgA converted plasminogen to plasmin with the following parameters: K(m) < or = 1.5 microM and k(cat) = 0.55 s(-)(1). The ability of proteolytic fragments of Sk(U) to activate plasminogen was investigated. Only two C-terminal segments (97-261 and 123-261), which both contain the beta-domain (126-261), were shown to be active. They initiated plasminogen activation in complex with plasmin, but not with plasminogen, and thereby exhibited functional similarity to the staphylokinase. The fusion protein His(6)-Sk(U) (i.e., Sk(U) with a small N-terminal tag) acted exclusively in complex with plasmin as well. These observations demonstrate that (1) the N-terminal alpha-domain, including a native N-terminus, was necessary for "virgin" activation of the associated plasminogen in the Sk(U)-PgA complex and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.

Asunto(s)

Plasminógeno/metabolismo , Estreptoquinasa/química , Estreptoquinasa/metabolismo , Animales , Sitios de Unión , Bovinos , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Factor Xa/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Streptococcus/enzimología

RESUMEN

A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF-BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.

Asunto(s)

Proteínas Portadoras/química , Glándulas Mamarias Animales/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Bovinos , Cromatografía en Agarosa , Cromatografía Líquida de Alta Presión , Clonación Molecular , Calostro/química , Cisteína/química , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Biblioteca de Genes , Glicosilación , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Mapeo Peptídico , Embarazo , Unión Proteica , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen Streptococcus uberis NCTC 3858. After the final reverse-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial peptide map was established, and degenerate primers were designed and used for amplification of fragments of the gene encoding the activator. Inverse PCR was subsequently used for obtaining the full-length gene. The S. uberis plasminogen activator gene (skc) encodes a protein consisting of 286 amino acids including a signal peptide of 25 amino acids. In an amino acid sequence comparison the cloned activator showed an identity of approximately 26% to the streptokinases isolated from Streptococcus equisimilis and Streptococcus pyogenes. Interestingly, the activator from S. uberis was found to lack the C-terminal domain possessed by the streptokinase from S. equisimilis. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of S. uberis revealed that 9 of these were highly similar to strain NCTC 3858. Sequencing of the skc gene from three of these strains indicated that the amino acid sequence of the protein is highly conserved within the species.

Asunto(s)

Activadores Plasminogénicos/aislamiento & purificación , Streptococcus/enzimología , Estreptoquinasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Vacunas Bacterianas/inmunología , Secuencia de Bases , Bovinos , Clonación Molecular , Datos de Secuencia Molecular , Activadores Plasminogénicos/genética , Streptococcus/inmunología , Estreptoquinasa/genética , Estreptoquinasa/inmunología

RESUMEN

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.

Asunto(s)

Plasminógeno/metabolismo , Proteínas Recombinantes/metabolismo , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , Animales , Catálisis , Bovinos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Productos de Degradación de Fibrina-Fibrinógeno/farmacología , Expresión Génica , Humanos , Cinética , Pichia/enzimología , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/aislamiento & purificación

RESUMEN

Proteose-peptone component 3 is a phosphorylated glycoprotein that was isolated from the proteose-peptone fraction of caprine milk. By mass spectrometric analysis, amino acid sequencing, and polymerase chain reaction analysis, the primary structure has been determined and has been shown to contain 136 amino acids. Phosphorylations were identified at Ser30 and Ser41. A partial glycosylation was present at Thr16, and a N-linked glycosylation was present at Asn78. Galactosamine was the amino sugar detected at Thr16. Glucosamine and galactosamine were the amino sugars found in the carbohydrate group linked to Asn78. The caprine amino acid sequence exhibits 88% identity with the bovine proteose-peptone component 3 sequence. However, when compared with the bovine sequence, the caprine sequence contains an insertion of a serine residue at position 25.

Asunto(s)

Caseínas/química , Glicoproteínas/química , Cabras , Leche/química , Fragmentos de Péptidos/química , Fosfoproteínas/química , Secuencia de Aminoácidos , Amino Azúcares/análisis , Animales , Conformación de Carbohidratos , Caseínas/metabolismo , Bovinos , Femenino , Glicoproteínas/metabolismo , Glicosilación , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Fosfoserina/análisis , Homología de Secuencia

RESUMEN

Proteose peptone component 3 (PP3) is a protein synthesized in the bovine mammary gland. A genomic 4.5-kb clone has been sequenced comprising a 2.9-kb PP3 transcriptional unit plus 1 kb of 5' and 0.6 kb of 3' flanking sequence. Bovine retroposons of the short interspersed nuclear element sequence class (SINES) and one microsatellite were localized in the PP3 gene. PP3 is homologous with the murine glycosylation-dependent cell-adhesion molecule 1 (GlyCAM 1) and this homology also extends to the exon/intron organization of the genes.

Asunto(s)

Adenosina Trifosfatasas/genética , Caseínas/genética , Moléculas de Adhesión Celular/genética , Glicoproteínas/genética , Fragmentos de Péptidos/genética , Animales , Antígenos CD , Secuencia de Bases , Bovinos , Genes , Ratones , Datos de Secuencia Molecular

RESUMEN

Here we report the molecular cloning and sequencing of three types of human alpha s1-casein transcripts and present evidence indicating that exon skipping is responsible for deleted mRNA transcripts. The largest transcript comprised 981 bp encoding a signal peptide of 15 amino acids followed by the mature alpha s1-casein sequence of 170 amino acids. Human alpha s1-casein has been reported to exist naturally as a multimer in complex with kappa-casein in mature human milk, thereby being unique among alpha s1-caseins [Rasmussen, Due and Petersen (1995) Comp. Biochem. Physiol., in the press]. The present demonstration of three cysteines in the mature protein provides a molecular explanation of the interactions in this complex. Tissue-specific expression of human alpha s1-casein was indicated by Northern-blot analysis. In addition, two cryptic exons were localized in the bovine alpha s1-casein gene.

Asunto(s)

Caseínas/genética , ARN Mensajero/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Caseínas/metabolismo , Clonación Molecular , ADN Complementario , Exones , Humanos , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Aminoácido

RESUMEN

A full length PP3 (Proteose-Peptone component 3) cDNA of 679 bp was isolated from a bovine mammary gland cDNA library. The cDNA encodes a signal peptide of 18 amino acids followed by the mature PP3 sequence of 135 amino acids. This polypeptide showed homology with mouse and rat GlyCAM-1 (Glycosylation dependent Cell Adhesion Molecule 1) a protein which has been shown to act as a ligand for lymphocytes. The similarity was most profound between the signal peptides and three short regions of the mature polypeptides. Additionally structural conservation was predicted by computer analysis in the shape of a C-terminal amphipathic helix. PP3 was found to be expressed in mammary gland but not in peripheral lymph nodes, Peyer's pathes, lung, spleen, heart, and muscle.

Asunto(s)

Caseínas/genética , Glicoproteínas/genética , Glándulas Mamarias Animales/metabolismo , Mucinas/genética , Fragmentos de Péptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario , Ratones , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido