The TSG-6 and IαI Interaction Promotes a Transesterification Cleaving the Protein-Glycosaminoglycan-Protein (PGP) Cross-link

Abstract

During co-incubation of human inter-␣-inhibitor (I␣I) and human tumor necrosis factor-stimulated gene 6 protein (TSG-6) SDS-stable interactions are formed between the two proteins. We have analyzed the products of this reaction and characterized the mechanism of complex formation. Following the incubation seven new bands not previously identified were apparent in SDS-PAGE. Three of these bands did not contain TSG-6, including heavy chain (HC)1⅐bikunin, HC2⅐bikunin, and free bikunin. In addition high molecular weight complexes composed of the same components as I␣I, including HC1, HC2, and bikunin, were formed. The formation of these complexes was prevented by the addition of hyaluronan. The cross-links stabilizing these complexes displaying properties similar to the protein-glycosaminoglycan-protein (PGP) cross-link. The TSG-6-containing SDS-stable complexes were composed of HC1⅐TSG-6 or HC2⅐TSG-6 exclusively. Both glycosylated and non-glycosylated TSG-6 participated in the complex formation. The HC⅐TSG-6 cross-links were different from the PGP cross-link and were determined to be ester bonds between the ␣-carbonyl of the C-terminal Asp of the heavy chain and most likely a hydroxyl group containing the TSG-6 residue. The mechanism involved cleaving the PGP cross-link of I␣I during a transesterification reaction. A TSG-6 hydroxyl group reacts with the ester bond between the ␣-carbonyl of the C-terminal Asp residues of HC1 or HC2 and carbon-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. An intermediate is formed resulting in a partitioning of the reaction between HC(1 or 2)⅐TSG-6 complexes and transfer of HC(1 or 2) to the chondroitin via competing pathways.

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

“…In-gel Digestion and Peptide Mass Fingerprinting AnalysisIn-gel digestion of proteins was performed essentially as described before (15,34). The tryptic peptides were purified on C18 stage tips (Proxeon Biosystems A/S) and eluted directly onto the matrix-assisted laser desorption/ionization target using 1 l of 70% acetonitrile containing 4 mg/ml ␣-cyano-4-hydroxycinnamic acid.…”

“…The reaction involves two divalent cation-dependent transesterifications and a covalent intermediate composed of and the HC to be transferred (14 -16). In the first reaction, the ester bond, between the C termini of the HCs and the CS chain in I␣I is cleaved, and a new transient ester bond is formed between the C termini of the HCs and Ser-28 (numbered according to the preprotein) in TSG-6 (15,17). In the second transesterification, the transient ester bond is cleaved and reformed between the C terminus of the HCs and HA (14).…”

“…HC2 is required during both transesterification reactions, but the molecular mechanism is currently unknown (16). In addition to HA, C-0-S and the CS chain of the bikunin proteins may also function as HC acceptors during this reaction (15,16,18). The attachment of HCs to the bikunin CS chain generates new proteins designated HMW bikunin proteins (15,16).…”

“…In addition to HA, C-0-S and the CS chain of the bikunin proteins may also function as HC acceptors during this reaction (15,16,18). The attachment of HCs to the bikunin CS chain generates new proteins designated HMW bikunin proteins (15,16). The physiological importance of TSG-6 and of the unique interactions between TSG-6, bikunin proteins, and HA have been underlined in many different studies (19 -24), particularly the importance during the formation of the HA-rich extracellular matrix surrounding the oocyte in mammals (25)(26)(27).…”

The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation

“…The transfer of ITIHs onto HA requires tumour necrosis factor a-induced protein 6 (TNFAIP6), also known as TNF-stimulated gene 6 (Jessen and Odum, 2003). TNFAIP6 forms a stable complex (Rugg et al, 2005) with ITIH and HA during the transesterification reaction (Sanggaard et al, 2005). This formation of ITIH-HA complexes is thought to play an important role in the stabilization of HA-rich extracellular matrices, with TNFAIP6 acting as an essential cofactor and catalyst (Rugg et al, 2005).…”