Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes

Sjöberg, Mathilda; Fries, Erik

doi:10.1016/0003-9861(92)90509-u

Cited by 52 publications

(41 citation statements)

References 26 publications

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“…The two proteins are separated by a dibasic sequence and pulse-chase experiments with isolated rat hepatocytes have shown that the precursor is cleaved just before it reaches the cell surface (19). The same type of experiment has also shown that the cleavage of proH3 (which is a prerequisite for the coupling of H3 to bikunin) occurs about 10 min before the bikunin precursor is cleaved (19). These observations suggest that the bikunin and H3 precursors are cleaved in different compartments: the secretory vesicles and the trans-Golgi/ trans-Golgi network, respectively.…”

Section: Fig 5 Mutations In C-terminal Extension Reduce Cellular CLmentioning

confidence: 99%

“…Using transfected COS-1 cells, we have previously shown that these two residues are absolutely essential for cleavage (18). The fact that there is no known protease that specifically cleaves at an Asp-Pro site and that the cleavage of the heavy chain precursors occurs earlier than the cleavage of proproteins with dibasic sequences (19) prompted us to study this reaction further. In the present paper we present evidence that the cleavage of the H3 precursor is autocatalytic.…”

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confidence: 99%

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The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

Thuveson

Fries

2000

Journal of Biological Chemistry

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Pre-␣-inhibitor is a plasma protein whose physiological function is still unknown, but in vitro studies suggest that it might be involved in inflammatory reactions. Pre-␣-inhibitor consists of a 25-and a 75-kDa polypeptide: bikunin and heavy chain 3 (H3), respectively. H3 is synthesized with a 30-kDa C-terminal extension, which is released in the Golgi complex through cleavage between an Asp and a Pro residue. We now provide evidence that this cleavage is triggered by the low pH in the late Golgi and occurs through an intramolecular process. First, incubation in vitro of the H3 precursor (proH3) at pH 6.0 or lower results in rapid cleavage of the protein. Second, the rate of the cleavage reaction does not depend on the concentration of proH3 and is not affected by the presence of various protease inhibitors. Third, raising the pH in organelles of cells producing proH3 abolishes cleavage during secretion. The amino acid residues near the cleavage site of proH3 differ from those of previously described self-cleaving proteins, indicating that the mechanisms of scission are different.

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Section: Fig 5 Mutations In C-terminal Extension Reduce Cellular CLmentioning

confidence: 99%

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confidence: 99%

The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

Thuveson

Fries

2000

Journal of Biological Chemistry

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“…Bikunin carries a 7-kDa chondroitin sulfate chain (4,5), which is linked to the ␣-carbons of the C-terminal amino acid residues of H1 and H2 through two ester bonds (6). Pulse-chase experiments with isolated hepatocytes have shown that the coupling of bikunin to the heavy chains occurs in the Golgi complex immediately after the chondroitin sulfate chain has been completed (7,8). Bikunin, which occurs in plasma also in free form (9), is a proteinase inhibitor with a wide specificity range but with relatively low affinity to the proteolytic enzymes tested so far (10).…”

Section: Inter-␣-inhibitor (I␣i)mentioning

confidence: 99%

Structural Characterization of Inter-α-inhibitor

Blom

Mörgelin

Öyen

et al. 1999

Journal of Biological Chemistry

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Inter-␣-inhibitor (I␣I) is a 180-kDa serum protein consisting of three polypeptides. Two of these, the heavy chains 1 and 2 (H1 and H2), are of 75-80 kDa and have similar amino acid sequences. The third polypeptide, bikunin, has a molecular mass of 25 kDa and contains a 7-kDa chondroitin sulfate chain that is covalently linked to the C-terminal amino acid residues of H1 and H2. I␣I has been shown to be required for the formation of the hyaluronan-containing extracellular matrix of certain cell types. How I␣I exerts this function is not known, but it appears that upon interaction with cells, the heavy chains are released and become covalently linked to hyaluronan. Our results indicate that I␣I and its heavy chains are extended molecules; thus, upon electron microscopy, I␣I appeared to consist of two globular domains connected by a thin structure 31-nm long and the isolated heavy chains of a globular domain and a "tail" about 15-nm long. Analysis of the heavy chains by partial proteolysis showed that the C-terminal halves are particularly sensitive to hydrolysis indicating that they are loosely folded. Furthermore, electron microscopy showed that partially degraded heavy chains lacked the extended regions. Taken together, these results suggest that the N-terminal half of the heavy chains forms a globular domain, whereas the other half has an extended and loosely folded structure.

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“…The rat homologue of ~-m was purified using rat urine from animals with kidney impairment [17] as starting material, or from the culture medium of transfected Hi 5 insect cells. The urine or cell culture medium was concentrated by ultrafiltration, and then subjected to affinity chromatography on a column containing monoclonal mouse anti-(rat and human ~-m) antibodies BN 11.10 [18], immobilized to Affi-gel Hz (40 mg/2 ml gel) according to instructions provided by the manufacturer (Bio-Rad Laboratories).…”

Section: Purification O/c~;-mmentioning

confidence: 99%

Formation of the α₁‐microglobulin chromophore in mammalian and insect cells: a novel post‐translational mechanism?

1995

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a~-Microglobulin is an immunosuppressive plasma protein synthesized by the liver. The isolated protein is yellowbrown, but the hypothetical chromophore has not yet been identified. In this work, it is shown that a human liver cell line, HepG2, grown in a completely synthetic and serum-free medium, secretes ~cmicroglobulin which is also yellow-brown, suggesting a de novo synthesis of the chromophore by the cells, at-Microglobulin isolated from the culture medium of insect cells transfected with the gene for rat a~-microglobulin is also yellow-brown, suggesting that the gene carries information about the chromophore. Reduction and alkylation or removal of N-or O-linked carbohydrates by glycosidase treatment did not reduce the colour intensity of the protein. An internal dodecapeptide (amino acid positions 70-81 in human aj-microglobulin) was also yellow-brown. The latter results indicate that the chromophore is linked to the polypeptide. in conclusion, the results suggest that the a~-microglobulin gene carries information activating a post-translational protein modification mechanism which is present in mammalian and insect cells.

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Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes

Cited by 52 publications

References 26 publications

The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

Structural Characterization of Inter-α-inhibitor

Formation of the α₁‐microglobulin chromophore in mammalian and insect cells: a novel post‐translational mechanism?

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Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes

Cited by 52 publications

References 26 publications

The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

Structural Characterization of Inter-α-inhibitor

Formation of the α1‐microglobulin chromophore in mammalian and insect cells: a novel post‐translational mechanism?

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Formation of the α₁‐microglobulin chromophore in mammalian and insect cells: a novel post‐translational mechanism?