<i>In vivo</i>Binding of Human Inter-α-Trypsin Inhibitor Free Heavy Chains to Hyaluronic Acid

Jessen, Torben E.; Ødum, Lars; Johnsen, Anders H.

doi:10.1515/bchm3.1994.375.8.521

Cited by 44 publications

(39 citation statements)

References 21 publications

(10 reference statements)

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“…6). An early study also found the retention of SHAP-HA complex when synovial fluids or follicle fluids were subjected to agarose gel electrophoresis (27). Our results were in agreement with the observation and further indicated that the SHAP-HA complex itself was enough to form the aggregates, which implies an interesting role for SHAP in the regulation of HA aggregation.…”

Section: Discussionsupporting

confidence: 90%

Molecular Heterogeneity of the SHAP-Hyaluronan Complex

Yingsung

Zhuo

Mörgelin

et al. 2003

Journal of Biological Chemistry

101

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We previously found that a covalent complex of SHAPs (serum-derived hyaluronan-associated proteins), the heavy chains of inter-␣-trypsin inhibitor family molecules, with hyaluronan (HA) is accumulated in synovial fluid of patients with rheumatoid arthritis, and the complex is circulated in patient plasma at high concentrations. How the SHAP-HA complex participates in this disease is unknown. To address this question, it is essential to clarify the structural features of this macromolecule. The SHAP-HA complex purified from synovial fluid of the patients by three sequential CsCl isopycnic centrifugations was heterogeneous in density, and the fractions with different densities had distinct SHAPto-HA ratios. Agarose gel electrophoresis and column chromatography revealed that there was no apparent difference in the size distribution of HA to which SHAPs were bound between the fractions with different densities. The SHAP-HA complex in the higher density fraction had fewer SHAP molecules per HA chain. Therefore, the difference between the fractions with different densities was due to a heterogeneous population of the SHAP-HA complex, namely the different number of SHAP molecules bound to an HA chain. Based on the SHAP and HA contents of the purified preparations, we estimated that an HA chain with a molecular weight of 2 ؋ 10 6 has as many as five covalently bound SHAPs, which could give a proteinaceous multivalency to HA. Furthermore, we also found that the SHAP-HA complex tends to form aggregates, judging from the migration and elution profiles in agarose gel electrophoresis and gel filtration, respectively. The multivalent feature of the SHAP-HA complex was also confirmed by the negative staining electron micrographic images of the purified fractions. Taken together, those structural characteristics may underlie the aggregate-forming and extracellular matrix-stabilizing ability of the SHAP-HA complex.

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Section: Discussionsupporting

confidence: 90%

Molecular Heterogeneity of the SHAP-Hyaluronan Complex

Yingsung

Zhuo

Mörgelin

et al. 2003

Journal of Biological Chemistry

101

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“…9,10) After the binding of the ITI family to hyaluronic acid, bikunin is released by an unknown mechanism and the heavy chains bind to hyaluronic acid covalently. [18][19][20] A feature of IHRP is its sensitivity to plasma kallikrein like kininogens.…”

Section: Quantitation Of Ihrp In Human Plasmamentioning

confidence: 99%

Quantitative Measurement of the Novel Human Plasma Protein, IHRP, by Sandwich ELISA.

Choi-Miura

2001

Biological & Pharmaceutical Bulletin

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We purified a 120-kDa novel glycoprotein from human plasma and named it IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) because of its sequence similarity to the heavy chains of the inter-alpha-trypsin inhibitor (ITI) family.1) IHRP was certified as the identical protein with PK-120 which was purified as a plasma kallikreinsensitive protein from human plasma.2) The ITI family includes ITI, pre-alpha-trypsin inhibitor and H2-bikunin. [3][4][5] All of them have the unique structures, i.e., bikunin and heavy chain(s) are bridged by a chondroitin sulfate sugar chain. [5][6][7][8] The function of the ITI family is believed to be as a protease inhibitor, however, the extracellular matrix-stabilizing activity of the ITI family is noteworthy. 9,10) In spite of the sequence similarity, IHRP did not form a complex with bikunin because of the deletion of the consensus sequence (DPHFII) for the cleavage of the C-terminal fragment and the attachment to the chondroitin sulfate sugar chain of bikunin.11,12) IHRP was readily cleaved to the N-terminal 85-kDa-fragment and the C-terminal 35-kDa fragment by plasma kallikrein. 1,2) The N-terminal 85-kDa fragment was further cleaved to the N-terminal 57-kDa fragment and to unidentified small fragments. 1,2) In a search of the nucleotide sequence homology of IHRPcDNA, we found that the nucleotide sequence registered as porcine ITI cDNA in LASL-GDB (Gen Bank) data base showed significant homology (83%) to that of IHRP cDNA.11) Since this sequence homology was higher than those (49-57%) of mRNAs among IHRP and ITI family heavy chains, we thought that the registered porcine ITI mRNA, which was detected as the inducible mRNA in the liver after resuscitation from cardiogenic shock, 13) was the species counterpart of human IHRP mRNA rather than porcine ITI family heavy chain mRNA.In this paper, we describe the preparation and the characterization of anti-IHRP mouse monoclonal antibodies and the quantitative measurement of the plasma IHRP concentrations of healthy donors and patients with inflammatory disorders. MATERIALS AND METHODSPreparation of Anti-IHRP Mouse Monoclonal Antibodies Anti-IHRP mouse monoclonal antibodies were prepared according to the method of Galfre et al. 14) A female Balb/c mice was immunized with 50 mg of purified human IHRP 1) as an emulsion with Freund's complete adjuvant. Three weeks after the first immunization, the mice were boosted with 50 mg of IHRP in phosphate-buffered saline (PBS). Three days after the boost injection, the spleen cells of the mice were fused with P3U1 mouse myeloma cells using polyethylene glycol (PEG). The hybridoma cells were selected in HAT medium and the media were screened by ELISA. The positive hybridoma cells were cloned by limiting dilution and the cloned cells (1ϫ10 7 cells/mice) were injected intraperitoneally into female Balb/c mice which had been pre-injected with pristane (0.5 ml/mice) one week earlier. Monoclonal antibodies were purified from the ascites fluids by ammonium precipitation and DEAE-Sephace...

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“…Thus hyaluronan isolated from the synovial fluid of patients with rheumatoid arthritis has been found to contain covalently linked heavy chains (19,20). Furthermore, upon inflammation, fibroblasts and monocytes secrete a protein named TSG-6, which reacts with I␣I by displacing H1 and forming a covalent link with the chondroitin sulfate of bikunin (21).…”

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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In vivoBinding of Human Inter-α-Trypsin Inhibitor Free Heavy Chains to Hyaluronic Acid

Cited by 44 publications

References 21 publications

Molecular Heterogeneity of the SHAP-Hyaluronan Complex

Molecular Heterogeneity of the SHAP-Hyaluronan Complex

Quantitative Measurement of the Novel Human Plasma Protein, IHRP, by Sandwich ELISA.

Structural Characterization of Inter-α-inhibitor

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