Rainer Deutzmann

Transient and locally restricted expression of laminin a chain mRNA by developing epithelial cells during kidney organogenesis

Three polypeptide chains, A, B1, and B2, have been described for mouse laminin, a basement membrane protein. We studied expression of laminin A, B1, and B2 mRNA in the developing mouse kidney. Induction of kidney mesenchyme differentiation in vitro led to an increased expression of B1 and B2 chain mRNA on day 1 of development. In contrast, expression of A chain mRNA increased on day 2, when epithelial cell polarization begins. Laminin A mRNA and polypeptide were expressed only by epithelia during in vivo development as well. Some polarized cell types producing basement membrane (endothelium, some adult epithelia) lacked the A chain mRNA and polypeptide, although they did express B chains. Laminin with the 400 kd A chain is therefore a transient form appearing at specific sites of kidney morphogenesis, whereas isoforms with a different A chain or without it have a more widespread distribution.

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.

Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 Å resolution

The bilin binding protein (BBP) from the insect Pieris brassicae has been analysed for amino acid sequence, spectral properties and three-dimensional structure. The crystal structure that had been determined by isomorphous replacement has been refined at 2.0 A (1 A = 0.1 nm) resolution to an R-value of 0.20. The asymmetric unit contains four independent subunits of BBP. The co-ordinate differences are 0.25 A, in accord with the estimated error in co-ordinates. The polypeptide chain fold is characterized by an eight-stranded barrel. The connecting loops splay out at the upper end of the barrel and open it, whilst the lower end is closed. The overall shape resembles a calyx. The biliverdin IX gamma chromophore is located in a central cleft at the upper end of the barrel. The bilatriene moiety is in cyclic helical geometry with configuration Z,Z,Z and conformation syn,syn,syn. The geometry is in accord with the spectral properties and permits a correlation between sign of the circular dichroism bands and sense of the bilatriene helices. The fold of BBP is related to retinol binding protein (RBP), as had been recognized in the preliminary analysis, although the amino acid sequences of RBP and BBP show only 10% homology. There are large differences in the loops at the upper end of the barrel, whilst the segments of the centre and the lower end of the barrel superimpose closely. The ligands of BBP and RBP, biliverdin and retinol, respectively, are also similarly located.

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)

Solubilization of protein BM‐40 from a basement membrane tumor with cheating agents and evidence for its identity with osteonectin and SPARC

Up to 80% of the calcium‐binding protein BM‐40 could be extracted from a tumor basement membrane with a physiological buffer containing 10 mM EDTA. About half of its amino acid sequence was determined by Edman degradation demonstrating identity with cDNA deduced sequences of bone osteonectin and SPARC.

Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes

A dissimilatory sulphite reductase was isolated from the extremely thermophilic dissimilatory sulphate-reducing archaeon Archaeoglobus fulgidus. In common with other dissimilatory sulphite reductases thus far characterized, the enzyme has an alpha 2 beta 2-structure and contains sirohaem, non-haem iron atoms and acid labile sulphide. The oxidized enzyme exhibited absorption maxima at 281, 394, 545 and 593 nm with a weak band around 715 nm. We have cloned and sequenced the genes for the alpha and beta subunits of this enzyme, which we designate dsrA and dsrB, respectively. They are contiguous in the order dsrA dsrB and probably comprise an operon, since dsrA is preceded by sequences characteristic of promoters in methanogenic archaea, and dsrB is followed by a sequence resembling termination signals in extremely thermophilic sulphur-dependent archaea. dsrA and dsrB encode 47.4 kDa and 41.7 kDa peptides, which have 25.6% amino acid sequence identity, indicating that they may have arisen by duplication of an ancestral gene. Each deduced peptide contains cysteine clusters resembling those postulated to bind sirohaem-[Fe4S4] complexes in sulphite reductases and nitrite reductases from other species. The dsrB encoded peptide lacks a single cysteine residue in one of the two clusters, suggesting that only the alpha subunit binds a sirohaem-[Fe4S4] complex, and chemical analyses showed the presence of only two sirohaems per alpha 2 beta 2 enzyme molecule. Both deduced peptides also contain an arrangement of cysteine residues characteristic of [Fe4S4] ferredoxins, and chemical analyses were consistent with the presence of six [Fe4S4] clusters per alpha 2 beta 2 enzyme molecule, two of which would be expected to be associated with sirohaem while the other four could bind to the ferredoxin-like sites.

Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin)

Amino acid sequences of 285 and 286 residues, respectively, were deduced for mouse and human BM‐40 from cDNA clones isolated from expression libraries. The sequences showed 92% identity and were also essentially identical to those of bone osteonectin and of the parietal endoderm protein SPARC. About 60% of the mouse BM‐40 sequence was confirmed by Edman degradation. Two of the seven disulfide bonds were localized which apparently separate two distinct domains of mouse BM‐40.

A dissimilatory sirohaem-sulfite-reductase-type protein from the hyperthermophilic archaeon Pyrobaculum islandicum

A sulfite-reductase-type protein was purified from the hyperthermophilic crenarchaeote Pyrobaculum islandicum grown chemoorganoheterotrophically with thiosulfate as terminal electron acceptor. In common with dissimilatory sulfite reductases the protein has an alpha 2 beta 2 structure and contains high-spin sirohaem, non-haem iron and acid-labile sulfide. The oxidized protein exhibits absorption maxima at 280, 392, 578 and 710 nm with shoulders at 430 and 610 nm. The isoelectric point of pH 8.4 sets the protein apart from all dissimilatory sulfite reductases characterized thus far. The genes for the alpha- and beta-subunits (dsrA and dsrB) are contiguous in the order dsrAdsrB and most probably comprise an operon with the directly following dsrG and dsrC genes. dsrG and dsrC encode products which are homologous to eukaryotic glutathione S-transferases and the proposed gamma-subunit of Desulfovibrio vulgaris sulfite reductase, respectively. dsrA and dsrB encode 44.2 kDa and 41.2 kDa peptides which show significant similarity to the two homologous subunits DsrA and DsrB of dissimilatory sulfite reductases. Phylogenetic analyses indicate a common protogenotic origin of the P. islandicum protein and the dissimilatory sulfite reductases from sulfate-reducing and sulfide-oxidizing prokaryotes. However, the protein from P. islandicum and the sulfite reductases from sulfate-reducers and from sulfur-oxidizers most probably evolved into three independent lineages prior to divergence of archaea and bacteria.

Structure and Biology of the Laminin-Nidogen Complex

A large variety of cells in the body are exposed to extracellular matrices which affect cell function and behavior. Basement membranes are prominent matrix structures and have been shown to consist of several collagenous and non-collagenous glycoproteins and proteoglycans (Martin and Timpl, 1987). Laminin is one of the major and ubiquitous components that was originally isolated and characterized from a tumor basement membrane as a cross-shaped, multidomain protein (Timpl et al., 1979; Engel et al., 1981). It is considered to be one of the important cell-binding proteins present in basement membranes and in addition possesses binding potentials for other matrix components. Another non-collagenous glycoprotein, nidogen/entactin, was initially discovered as a sulfated 158kD polypeptide (Carlin et al., 1981) and shown to have a characteristic dumb-bell structure (Paulsson et al., 1986; 1987). Even though nidogen is also an ubiquitous constituent of basement membranes its function is not immediately apparent. However, several pieces of indirect evidence from cell culture and tissue studies (Dziadek and Timpl, 1985) provided initial clues about the possible role of nidogen, by showing that it forms tight, stoichiometric, noncovalent complexes with laminin in situ.