Karlheinz Mann

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd‐I (positions 1–905) comprising the N‐terminal globule and rod‐like domain and Nd‐II corresponding mainly to the C‐terminal globule (position 906–1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N‐terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N‐terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31–56 kDa) connected by either a rod‐like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd‐I binding to collagen IV and fragment Nd‐II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd‐I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C‐terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non‐covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25–32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N‐terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin‐1 and fibulin‐2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin‐1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22–38 kDa) and in serum (120–300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N‐terminal association region (∼50 residues), a central protease‐sensitive hinge region (∼70 residues) and a C‐terminal stable endostatin domain (∼180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix‐associated to a more soluble endocrine form.

A single EGF-like motif of laminin is responsible for high affinity nidogen binding.

A major nidogen binding site of mouse laminin was previously localized to about three EGF‐like repeats (Nos 3–5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3‐5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3‐5 and nidogen could be efficiently converted into a covalent complex by cross‐linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3‐5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3‐5 was indicated from its protease resistance and the complete loss of cross‐reacting epitopes as well as of nidogen‐binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF‐like repeats 3–4 and 4–5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen‐binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Abstract Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μ M ) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ .

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28‐residue signal peptide. The data support a previously proposed dumb‐bell model of nidogen by demonstrating a large N‐terminal globular domain (641 residues), five EGF‐like repeats constituting the rod‐like domain (248 residues) and a smaller C‐terminal globule (328 residues). Two more EGF‐like repeats interrupt the N‐terminal and terminate the C‐terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta‐hydroxylation, two N‐linked carbohydrate acceptor sites and, within one of the EGF‐like repeats an Arg‐Gly‐Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C‐terminal globule but not yet precisely localized.

Confirmation of the existence of a third family among peptidyl‐prolyl cis/trans isomerases Amino acid sequence and recombinant production of parvulin

In addition to the major cyclophilin‐like peptidyl‐prolyl cis/trans isomerases (PPIases) of Escherichia coli an enzyme of very low relative molecular mass (10.1 kDa) was discovered in this organism which gave first indication of the existence of a novel family in this enzyme class [1994, FEBS Lett. 343, 65–69]. In the present report we describe the chemically determined amino acid sequence of four peptides derived from the 10.1 kDa protein by the treatment with either cyanogen bromide or endoproteinase Lys‐C. Together with a continuous run of 75 amino acids starting N‐terminally, the sequence of the mature enzyme, 92 residues in length, was elucidated. Cloning and determination of the primary structure of a DNA fragment encoding this enzyme were also performed. Overexpression of the enzyme by using multicopies of plasmid pSEP38 in E. coli and detecting an enhanced PPIase activity attributed to the 10.1 kDa enzyme provided additional proof that the 92 amino acid protein was a PPIase. The enzyme was called parvulin (lat.: parvulus, very small). Homology analyses indicated that several parvulin‐like proteins could be found in the database screened. To further elucidate the functional role of PPIases it might be of some importance that homologous proteins like the PrtM protein of Lactococcus lactis and the PrsA lipoprotein of Bacillus subtilis are known to be involved in the protein export and maturation machinery of the bacteria.

Mosaic structure of globular domains in the human type VI collagen alpha 3 chain: similarity to von Willebrand factor, fibronectin, actin, salivary proteins and aprotinin type protease inhibitors.

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N‐terminal globule (1804 residues), a central triple helical segment (336 residues) and a C‐terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N‐terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17‐25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C‐terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C‐terminal domain C5 (70 residues) shows 40‐50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg‐Gly‐Asp cell‐binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)

The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1‐F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C‐terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N‐terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2‐alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity.

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macro‐phage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506‐binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl‐prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K‐12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP‐type PPlases of eukaryotes. However, the N‐terminal extension of Mip and/or the substitutions of the variable amino acids in the C‐terminal FKBP core lead to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein‐folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.

Effective activation of the proenzyme form of the urokinase‐type plasminogen activator (pro‐uPA) by the cysteine protease cathepsin L

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase‐type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single‐chain proenzyme (pro‐uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro‐uPA. Enzymatic assays, SDS‐PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro‐uPA. As determined by N‐terminal amino acid sequence analysis, activation of pro‐uPA by cathepsin L is achieved by cleavage or the Lys158‐lle159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147‐5152) cleavage of pro‐uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro‐uPA of 1:2,000). Nevertheless, even at pH 7.0, pro‐uPA was activated by cathepsin L, although a 10‐fold higher concentration of cathepsin L was required. As tumor cells may produce both pro‐uPA and cathepsin L, implications for the activation of tumor cell‐derived pro‐uPA by cathepsin L may be considered. Different pathways activation of pro‐uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro‐uPA; (ii) activation by serine proteases (plasmin, kallikrein. Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).