The Transfer of Heavy Chains from Bikunin Proteins to Hyaluronan Requires Both TSG-6 and HC2

Kristian W Sanggaard,Carsten S Sonne-Schmidt,Toke P Krogager,Karen A Lorentzen,Hans-Georg Wisniewski,Ida B Thøgersen,Jan J Enghild

doi:10.1074/jbc.m800874200

Kristian W Sanggaard, Carsten S Sonne-Schmidt + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m800874200

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Abstract

Tumor necrosis factor-stimulated gene-6 protein (TSG-6) is involved in the transfer of heavy chains (HCs) from inter-alpha-inhibitor (IalphaI), pre-alpha-inhibitor, and as shown here HC2.bikunin to hyaluronan through the formation of covalent HC.TSG-6 intermediates. In contrast to IalphaI and HC2.bikunin, pre-alpha-inhibitor does not form a covalent complex in vitro using purified proteins but needs the presence of another factor (Rugg, M. S., Willis, A. C., Mukhopadhyay, D., Hascall, V. C., Fries, E., Fülöp, C., Milner, C. M., and Day, A. J. (2005) J. Biol. Chem. 280, 25674-25686). In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including IalphaI, HC2.bikunin, and free HC2 promoted the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur.

Highlights

Only three of the five homologous heavy chains (HCs) have been identified in complex with bikunin
The results revealed that HC2 derived from HC21⁄7bikunin was transferred to HA (Fig. 4B, lane 7, band b) and that the HC21⁄7HA crosslink was dissociated by mild NaOH
The present study confirms that Tumor necrosis factor-stimulated gene-6 protein (TSG-6) alone is unable to transfer HC3 from P␣I to HA in vitro [15]. This observation has been puzzling in view of the fact that HC31⁄7HA has been observed in vivo [16], and studies with TSG-6 knock-out mice show that TSG-6 transfers HC1 and HC2 from I␣I and HC3 from P␣I to HA in vivo [26]

Summary

EXPERIMENTAL PROCEDURES

Materials—Magnesium chloride, HA sodium salt from human umbilical cord, and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Sigma-Aldrich. Complex formation was evaluated by reducing SDS-PAGE and immunoblotting using anti-TSG-6 antibodies Similar to this approach we treated HC21⁄7bikunin with NaOH, as described before [7], to generate free HC2. The tryptic pep- were incubated and analyzed by reducing SDS-PAGE and tides were purified on a C18 Stagetip (Proxeon Biosystems A/S), immunoblotting using an anti-TSG-6 antibody. Both I␣I and and eluted directly onto the MALDI target using 1 ␮l of matrix HC21⁄7bikunin promoted the formation of HC31⁄7TSG-6 com-. Significant hits as defined by Mascot proba- treated sample and the chondroitinase ABC-treated I␣I and bility analysis were accepted

RESULTS

Band a b c

IαI e

Findings

DISCUSSION