Tetranectin, a trimeric plasminogen-binding C-type lectin.

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.

Collagen-binding recombinant fibronectin fragments containing type II domains.

Each of the two type II domains and four larger fragments, containing one or two type II domains of fibronectin, have been expressed in Escherichia coli. A special vector, containing a fragment encoding the cleavage site for Factor Xa, Ile-Glu-Gly-Arg, inserted immediately before the protein fragment of interest, was used. After treatment of the purified fusion proteins with reduced/oxidized glutathione, the correctly folded fibronectin fragments were released by proteolytic digestion with Factor Xa. The largest fragment, consisting of two type II and two type I domains, was the only fragment able to bind to immobilized gelatin.

Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil.

Tetranectin is a plasminogen kringle 4-binding protein. The crystal structure has been determined at 2.8 A resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple alpha-helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected to a long alpha-helix. Tetranectin has been classified in a distinct group of the C-type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C-type lectin superfamily, whereas the third is present only in long-form CRDs. Tetranectin represents the first structure of a long-form CRD with intact calcium-binding sites. In tetranectin, the third disulfide bridge tethers the CRD to the long helix in the coiled coil. The trimerization of tetranectin as well as the fixation of the CRDs relative to the helices in the coiled coil indicate a demand for high specificity in the recognition and binding of ligands.

Receptor-binding domain of human alpha 2-macroglobulin. Expression, folding and biochemical characterization of a high-affinity recombinant derivative.

A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M. RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric. Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule.

Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein.

The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass--i.e., approximate sequence positions--that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1-177 and 94-323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94-177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro115-Asp177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.

Mass spectrometric characterisation of post-translational modification and genetic variation in human tetranectin.

Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein primarily involved in tissue remodeling and development, was scanned for covalent modifications and sequence heterogeneity, using a combination of mass spectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of different mass and abundance in plasma tetranectin, all of higher mass than that calculated from the cDNA sequence. To identify and locate residues accounting for the heterogeneity, samples of tetranectin were subjected to proteolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry and, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due to sequence heterogeneity at position 85 and the presence of a partially sialylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is encoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixture of Ser85 and Gly-85 sequence variants. Mass spectrometric analysis of enzymatic and mild acid hydrolysates of an N-terminal glycopeptide showed that the composition and partial covalent structure of the O-linked oligosaccharide prosthetic group is < or =N-acetylhexosamine < or =[hexose, (sialic acid)0-3].

Dissection of the domain architecture of the alpha2macroglobulin-receptor-associated protein.

The alpha2macroglobulin-receptor-associated protein (RAP) binds to the alpha2macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP), a multi-functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known alpha2MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands. In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-3415], to three structural domains, 1, 2 and 3, comprising residues 18-112, 113-218 and 219-323 of RAP, respectively. Structural analysis by 1H-NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of alpha-helices, whereas domain 3 as a separate domain appears only to be marginally stable. Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I-RAP and 125I-alpha2M* (methylamine-activated alpha2M) for binding to alpha2MR/LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3.

Protein-ligand interactions in the lysine-binding site of plasminogen kringle 4 are different in crystal and solution. Electrostatic interactions studied by site-directed mutagenesis exclude Lys35 as an important acceptor in solution.

Three amino acid residues previously reported to establish the interactions between lysine-like derivatives and plasminogen kringle 4 have been replaced by other residue types using the methods of site-directed mutagenesis. The effect of these modifications on the binding constant have been measured. The residues are Lys35, Asp57, and Arg71, according to the sequence numbering scheme adapted from the plasminogen kringle 5 domain. The plasminogen kringle 4 derivatives where Lys35 of the native molecule is replaced with isoleucine and methionine residues, respectively, were seen to bind the ligands, respectively, with association constants similar to those of the unmodified recombinant kringle 4 domain. The modification of Asp57 to asparagine was shown to eliminate the ability to bind to the lysine affinity column used to purify the protein. Similarly the site-directed mutagenesis for Arg71 to glutamine resulted in a 12-19-fold decrease in binding of each of the two ligands. In addition, the effect of ionic strength on the binding of 6-aminohexanoic acid to the recombinant plasminogen kringle 4 and the three single substituted derivatives was examined. For the unmodified kringle domain as well as for the two derivatives modified only at the position of Lys35, an ionic strength of 0.5 M reduced the binding constant by a factor of 3 to 0.12 x 10(5) M-1. The derivative modified at the position of Arg71 was not effected by the ionic strength and maintained a rather low binding constant of 0.02 x 10(5) M-1. The observations suggest that the carboxylate of Asp57 and the guanidino group of Arg71 provide the electrostatic interaction in the binding site for the epsilon-amino group and the alpha-carboxylate of a C-terminal lysine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and expression in Escherichia coli of the glycoprotein gene of VHS virus, and immunization of rainbow trout with the recombinant protein.

The gene encoding the envelope glycoprotein of a recent Danish isolate of a salmonid rhabdovirus, viral haemorrhagic septicaemia virus (VHSV) has been cloned and sequenced at the cDNA level. When compared with the deduced sequence of a French isolate of VHSV, it was noted that there were 13 amino acid substitutions in the Danish virus. Amino acid homologies with the glycoprotein of a North American salmonid rhabdovirus (infectious haematopoietic necrosis virus) indicate a high degree of structural similarity between the two fish rhabdovirus glycoproteins. Results from partial enzymatic deglycosylation of the viral protein indicate that all four NXT/S sites found in the sequence are N-glycosylated in the virus. The glycoprotein, without the N-terminal leader sequence and C-terminal hydrophobic anchor segment, was expressed in Escherichia coli as a factor Xa protease-cleavable fusion protein. The purified and renatured viral part of the recombinant protein was able to elicit VHSV-specific antibodies and neutralizing antibody activity in serum when injected into rainbow trout.

Human plasminogen binding protein tetranectin: crystallization and preliminary X-ray analysis of the C-type lectin CRD and the full-length protein.

The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates that trimers of TN are formed in accordance with the observation of trimerization in solution.

Alpha 2-macroglobulin-proteinase complexes, plasminogen activator inhibitor type-1-plasminogen activator complexes, and receptor-associated protein bind to a region of the alpha 2-macroglobulin receptor containing a cluster of eight complement-type repeats.

A region containing sites for ligand binding was localized in the 4525-amino acid residue alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) by ligand- and immunoblotting of proteinase and CNBr digests of the purified human placental protein. 125I-Labeled rat alpha 1-macroglobulin light chain, urokinase-plasminogen activator inhibitor type-1 complex, and alpha 2MR-associated protein all bound to a 75-kDa CNBr-generated fragment (68 kDa after deglycosylation). In addition to the three ligands, the fragment bound a novel monoclonal antibody reacting in the region defined by amino acid residues 1165-1246 as determined by binding to recombinant fragments of alpha 2MR/LRP. The positions of methionine residues in alpha 2MR/LRP suggested that the ligand-binding CNBr fragment contained three disulfide-linked peptides comprising the residues 776-1399. This origin was confirmed by partial amino acid sequencing of the electroblotted fragment and polypeptides generated by reduction of the fragment. The identified region represents 13.6% of the molecular mass (nonglycosylated) of alpha 2MR/LRP and contains one of three large clusters of complement-type repeats.

Identification of residues in alpha-macroglobulins important for binding to the alpha2-macroglobulin receptor/Low density lipoprotein receptor-related protein.

Variants of the receptor binding domain of both human alpha2-macroglobulin and the corresponding domain of hen egg white ovomacroglobulin have been expressed in Escherichia coli and refolded in vitro. Competition experiments with methylamine-treated alpha2-macroglobulin for binding to the multifunctional alpha2-macroglobulin receptor identify two Lys residues (residues 1370 and 1374 in human alpha2-macroglobulin) spaced by three amino acid residues as crucial for receptor binding. From this result and mutational evidence from other ligands for the alpha2-macroglobulin receptor, a tentative sequence motif for receptor binding is proposed.

Structure of the C-type lectin carbohydrate recognition domain of human tetranectin.

Tetranectin (TN) is a C-type lectin involved in fibrinolysis, being the only endogenous ligand known to bind specifically to the kringle 4 domain of plasminogen. TN was originally isolated from plasma, but shows a wide tissue distribution. Furthermore, TN has been found in the extracellular matrix of certain human carcinomas, whereas none or little is present in the corresponding normal tissue. The crystal structure of full-length trimeric TN (2.8 A resolution) has recently been published [Nielsen et al. (1997). FEBS Lett. 412, 388-396]. The crystal structure of the carbohydrate recognition domain (CRD) of human TN (TN3) has been determined separately at 2.0 A resolution in order to obtain detailed information on the two calcium binding sites. This information is essential for the elucidation of the specificity of TN towards oligosaccharides. TN3 crystallizes as a dimer, whereas it appears as a monomer in solution. The overall fold of TN3 is similar to other known CRDs. Each monomer is built of two distinct regions, one region consisting of six beta-strands and two alpha-helices, and the other region is composed of four loops harboring two calcium ions. The calcium ion at site 1 forms an eightfold coordinated complex and has Asp116, Glu120, Gly147, Glu150, Asn151, and one water molecule as ligands. The calcium ion at site 2, which is believed to be involved in recognition and binding of oligosaccharides, is sevenfold coordinated with ligands Gln143, Asp145, Glu150, Asp165, and two water molecules. One sulfate ion has been located at the surface of TN3, forming contacts to Glu120, Lys148, Asn106 of a symmetry-related molecule, and to an ethanol molecule.

Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.