Tetranectin, a trimeric plasminogen‐binding C‐type lectin

Abstract

Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization.Keywords: C-lectin; domain structure; refolding; trimerization Tetranectin (TN) is a homo-oligomeric Caz+-binding protein primarily found in plasma (10 mg/L) and in the extracellular matrix of certain human carcinomas, whereas little or no TN is found in the extracellular matrix of the corresponding normal tissues (Christensen & Clemmensen, 1989;Wewer &Albrechtsen, 1992). Although specific binding of TN to plasminogen kringle-4 domain (PLM K4), to complex sulfated polysaccharides (Clemmensen, 1989) and to proteins in the extracellular matrix have been shown (Kluft et al., 1989a(Kluft et al., , 1989b, indicating that TN may participate in processes involved in tissue remodeling, the physiological role of TN remains to be clarified.The mature TN polypeptide chain, 18 1 amino acid residues with three intra-chain disulfide bridges (Fuhlendorff et al., 1987), is encoded in three exons as shown by molecular cloning and characterization of the gene (Berglund & Petersen, 1992;Wewer & Albrechtsen, 1992). The posttranslational modification of threonine-4, originally reported by Fuhlendorff et al. (1987), has been identified as 0-linked glycosylation (Jaquinod et al., in prep.). On the basis of gel filtration data, natural human TN was originally proposed to occur as a homo-tetramer in solution (Clemmensen et al., 1986).Exon 3 of the human TN gene encodes a separate functional and structural unit, a single long-form carbohydrate recognition domain (CRD) clearly related to C-type lectins by sequence homology, conservation of disulfide topology (Fuhlendorff et al., 1987) and by presence of an almost complete suit of amino acid residues predicted to be involved in Ca2+ and sugar binding. Due to the Reprint requests to: Michael Etzerodt, Laboratory of Gene Expression, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark; e-mail: met@biobase.dk.'Deceased.reported tetrameric nature along with single residue differences TN is considered to belong to a distinct class of C-type lectins (Drickamer & Taylor, 1993;Day, 1994).In the present study, we report on cross-linking analysis of the homo-oligomeric organization of natural and recombinant TN in solution. ResultsA series of TN derivatives, rTN123 (El-V181, mature protein), rTN12 (El-V49, exon 1 and 2), rTN23 (V17-V181, exon 2 and 3), and rTN3 (A45-Vl8 1, exon 3), was expressed in E. coli and refolded in vitro with the purpose of analyzing the oligomeric nature...

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain

“…The recombinant TN (rTN) variants studied were identical with those described in [1], except that the construct representing the polypeptide encoded by exons 1 and 2 (rTRIP-A) corresponded to amino acid residues 1-54 and with Cys-50 replaced by Ser, whereas the rTN12 derivative described earlier [1] encoded residues 1-49. rTN3 corresponded to residues 45-181 (exon 3), encoding the CRD of TN. rTN23 corresponded to residues 17-181 (exons 2 and 3).…”

“…The chimaeric rM1TN construct was prepared by replacing the human TN sequence from Glu-1 to Leu-26 with the corresponding murine TN sequence [28], using the shared SacI restriction enzyme site (see Figure 4). Expression in Escherichia coli and purification of the unfolded rTN fusion protein derivatives were performed as described [1]. Refolding of the rTN, rTN23, rTN3 and rM1TN derivatives were performed by loading the fusion proteins on Ni# + -charged nitrilotriacetic acid (NTA) columns in 8 M urea\0.5 M NaCl\50 mM Tris\HCl (pH 8)\10 mM 2-mercaptoethanol.…”

“…This is demonstrated by the fact that neither rTN3 nor rTN23 was found to bind heparin and that the interaction strength for TN was not affected by Ca# + ions or EDTA. rTN23 is a trimer [1] and it would therefore be expected that a weak affinity site in the CRD, which might have been masked by a stronger interaction with the N-terminal binding site, should become apparent, because trimerization would generate an additive effect by presenting a high local concentration of such binding sites at the heparin-Sepharose surface.…”

“…Tetranectin (TN) is a homotrimeric [1] Ca# + -and plasminogen (Plg)-binding protein found in plasma [2] and as a component of the extracellular matrix during muscle development and regeneration [3]. TN has been proposed to have a role in mineralization during osteogenesis [4,5].…”

“…The mature TN monomer, of 181 amino acid residues and containing three internal disulphide bridges, is encoded by three exons [8,12], of which the polypeptide encoded by exon 2 is necessary and sufficient for trimerization [1]. The Plg kringle-4-binding site is located within the domain encoded by exon 3 [13], which encodes a domain similar with the carbohydrate recognition domains (CRDs) of the C-type lectin superfamily [14].…”