One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate

Sjöberg, Mathilda; Fries, Erik

doi:10.1042/bj2720113

Cited by 15 publications

(9 citation statements)

References 29 publications

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“…In addition, bikunin has been shown to be capable of neutralizing granzyme K (40). Granzymes, along with perforins, are major components of cytosolic granules that play an important role in T-cell-and natural killer cell-mediated cytotoxicity against virally infected host cells (6,30,31). Other pharmaceutical effects of bikunin, like suppression of pancreatitis, colitis, and arthritis, also point towards bikunin as an immunosuppressive protein (11).…”

Section: Hismentioning

confidence: 99%

“…In urine it exists in free form, while in blood serum both free and I␣I-complexed forms are present (28). Bikunin belongs to the Kunitz family of protease inhibitors, by virtue of two tandemly arranged Kunitztype domains, and is active against a broad range of enzymes that include trypsin, chymotrypsin, plasmin, leukocyte elastase, cathepsin B, and cathepsin H (9,16,31). Bikunin provides the I␣I family members with their protease inhibitory capacity, and the I␣I family has been implicated in inflammatory responses (28).…”

Section: Hismentioning

confidence: 99%

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The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Tyagi

Surjit

Lal

2005

J Virol

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Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF).Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (␣ 1 -microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of ␣ 1 -microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His 6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where it causes epidemics and sporadic infections. The causative agent, hepatitis E virus (HEV), is transmitted via the fecal-oral route, predominantly through contaminated water (7,10,25,26). HEV is a plusstranded RNA virus with a 7.2-kb genome containing three open reading frames (ORF), ORF1, ORF2, and ORF3, encoding three different proteins (20,32,35). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for the putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,20,27,35). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus, and yeast and in vitro in a coupled transcription-translation system (12,15,17,22,23). ORF2, the major capsid protein for HEV, is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of selfassociation (18, 36). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton, and shows no major processing (3, 42). The ORF3 protein dimerizes using a 43-amino-acid region, interacts with SH3 domains, and activates mitogen-activated protein kinase (21, 37). The phosphorylated form of the ORF3 protein has also been shown to interact with the nonglycosylated form of the major capsid protein, ORF2 (38). These properties of ORF3 clearly indicate that this protein may have multiple roles in HEV pathogenesis. Rece...

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Section: Hismentioning

confidence: 99%

Section: Hismentioning

confidence: 99%

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Tyagi

Surjit

Lal

2005

J Virol

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“…a sulphated, heterogeneous glycosaminoglycan to the bikunin peptide [26]. Transfection of COS cells with DNA encoding a,-microglobulin-bikunin with ot without furin or bikunin alone yielded a similar sulphated and heterogeneous molecule, most likely free bikunin (Fig.…”

Section: Rpe Chomentioning

confidence: 99%

Processing and secretion of rat α₁‐microglobulin‐bikunin expressed in eukaryotic cell lines

1994

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The precursor protein a,-microglobulin-bikunin was cleaved to the same degree whether expressed in CHO cells or in mutated CHO cells, RPE.40 cells, suggested to lack a functional form of the intracellular protease furin. Thus, a,-microglobulin-bikunin probably is not cleaved in vivo by furin. However, simultaneous overexpression of the precursor and furin in COS, CHO and RPE.40 cells increased the cleavage, suggesting that compartmentalisation and concentrations of protease and precursor are important for the cleavage, besides the in vitro specificity. Expression of a,-microglobulin and bikunin alone gave different protein patterns on SDS-PAGE as compared to expression of the precursor and subsequent cleavage, suggesting that the precursor protein is important for the post-translational handling of a,-microglobulin and bikunin.

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“…H3 has so far been shown to be synthesized only by hepatocytes (21,22). In these cells, bikunin is by far the most abundant chondroitin sulfate-containing protein (11,23), which could explain why only this protein has been found to be linked to H3. Perhaps more sensitive detection techniques would reveal that hepatocytes secrete other heavy chain-linked proteins.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Coupling of the Heavy Chain of Pre-α-inhibitor to Chondroitin Sulfate

Kaczmarczyk

Thuveson

Fries

2002

Journal of Biological Chemistry

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Pre-␣-inhibitor is a serum protein consisting of two polypeptides, the heavy chain and bikunin, covalently linked through an ester bond between the chondroitin sulfate chain of bikunin and the ␣-carboxyl group of the carboxyl-terminal residue of the heavy chain. The heavy chain is synthesized with a carboxyl-terminal extension, which is cleaved off just before the link to bikunin is formed. Our earlier studies indicate that this extension mediates the cleavage, and we have now found that a short segment on the amino-terminal side of the cleavage site is also required for the reaction. Furthermore, we previously showed that coexpression of the heavy chain precursor and bikunin in COS-1 cells leads to linkage, and we have now used this system to identify a His residue in the carboxyl-terminal extension that is specifically required for the intracellular coupling of the two proteins. In addition, we have shown that another chondroitin sulfate-containing protein, decorin, will also form a complex with the heavy chain, as will free chondroitin sulfate chains. These results suggest that in vivo there might be other, as yet unknown, chondroitin sulfate-containing polypeptides linked to the heavy chain.

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One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate

Cited by 15 publications

References 29 publications

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Processing and secretion of rat α₁‐microglobulin‐bikunin expressed in eukaryotic cell lines

Intracellular Coupling of the Heavy Chain of Pre-α-inhibitor to Chondroitin Sulfate

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One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate

Cited by 15 publications

References 29 publications

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Processing and secretion of rat α1‐microglobulin‐bikunin expressed in eukaryotic cell lines

Intracellular Coupling of the Heavy Chain of Pre-α-inhibitor to Chondroitin Sulfate

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Processing and secretion of rat α₁‐microglobulin‐bikunin expressed in eukaryotic cell lines