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K Kühn

Publications and source records attributed to K Kühn.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cooperative and competitive interactions of regulatory elements are involved in the control of divergent transcription of human Col4A1 and Col4A2 genes.

The genes COL4A1 and COL4A2, coding for the two subunit chains alpha1(IV) and alpha2(IV) of collagen IV [alpha1(IV)2alpha2(IV)] are found closely linked on the human chromosome 13 in a unique head-to-head arrangement resulting in opposite strand transcription starting from a shared promoter region. Transient transfection experiments defined a shared promoter and two symmetrically arranged, downstream located and gene-specific activating elements in each gene. The shared promoter does not exhibit any transcriptional activity and efficient transcription depends on the cooperative effect of downstream elements. Mutual inhibitory effects between the two activating elements indicate competitive interactions with the shared promoter. Symmetry, cooperativity and competitivity of cis-elements are also reflected by the binding of transacting factors to the promoter and activating elements. From these data we propose a model for the coordination of divergent transcription of COL4 genes based on the cooperative and competitive interactions of the shared promoter and gene-specific regulating elements.

Binding, Competitive↗
PubMed1997

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Antibodies, Monoclonal↗
PubMed1996

The recognition sites of the integrins alpha1beta1 and alpha2beta1 within collagen IV are protected against gelatinase A attack in the native protein.

The susceptibility of three different solubilized forms of type IV collagen to gelatinase A cleavage and the concomitant effects on cell and integrin binding have been assessed. Dithiothreitol-solubilized Engelbreth-Holm Swarm (EHS) type IV collagen with disrupted intramolecular disulfide bonds in the CB3[IV] region was cleaved N-terminally to the CB3[IV] region into the two characteristic 100-300-nm fragments at 30 degrees C and was totally degraded at 37 degrees C. This was reflected in the partial or total loss of the alpha1beta1 and alpha2beta1 integrin binding sites within this region. The ability of gelatinase A to cleave EHS type IV collagen preparations with intact interchain disulfide bonds in CB3[IV] only occurred at higher temperatures. Furthermore, no effect on binding of cells or isolated integrins to the gelatinase-treated collagen could be detected after treatment at 37 degrees C. Dimeric collagen IV of human placenta with intact disulfide bonds in the CB3[IV] region was not degraded at all by gelatinase A at 37 degrees C.

Amino Acid Sequence↗
PubMed1996

Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. Models for collagen catabolism by matrix-metalloproteases.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-P'9/10) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)5 triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple-stranded cystine peptide. While the single chains showed no tendency to self-association into triple helices, the heterotrimer (alpha1 alpha2 alpha1') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tm) of 33 degrees C. This triple-helical collagen-like peptide is cleaved by the full-length human neutrophil collagenase (MMP-8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single alpha1' chain. In contrast, opposite digestion rates were measured with the catalytic Phe79-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits Km values of 5 microM and 1 mM for the heterotrimer and the single alpha1' chain, respectively, which compare well with those reported for collagen type I (approximately 1 microM), gelatine (approximately 10 microM) and for octapeptides of the cleavage sequence (> or = 1 mM). The high affinity of the MMP-8 for the triple-helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

Amino Acid Sequence↗
PubMed1996

Identification of the endothelial cell binding site for factor IX.

We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined.

Animals↗
PubMed1995

The function of the NC1 domains in type IV collagen.

At its C terminus, the collagen IV molecule bears a globular NC1 domain, to which two functions have been assigned. In the macromolecular network of collagen IV, two molecules are connected via their NC1 domains, which form a hexameric complex, stabilized by intermolecular disulfide bonds. In addition, the NC1 domains are thought to be responsible for chain selection and assembly. In order to understand the role of the NC1 domains during these steps, hexameric complexes were isolated and further investigated. SDS-polyacrylamide gel electrophoresis and Western blot revealed disulfide-linked alpha 1 (IV)NC1 and alpha 2(IV)NC1 homodimers but no heterodimers. The hexamers were dissociated at low pH, separated into monomers and dimers, and submitted to reconstitution experiments. Only alpha 1(IV)NC1 dimers were able to reconstitute a hexameric complex. alpha(IV)-NC1 and alpha 2(IV)NC1 monomers as well as the alpha 2(IV)NC1 dimers showed only a low tendency to form complexes. It is assumed that during formation of the collagen IV network, lateral aggregation of the molecules via the triple helical domains brings the C termini of two molecules into close vicinity and that subsequently the weak interactions observed between the NC1 subdomains provide the correct alignment for a disulfide exchange. It is, however, questionable whether the low affinity between the NC1 subdomains alone is sufficient for chain assembly and alignment of the alpha(IV) chains before molecule formation.

Collagen↗
PubMed1995

Identification and characterization of a novel transcriptional silencer in the human collagen type IV gene COL4A2.

Collagen type IV [alpha 1(IV)2 alpha 2(IV)] is the basic structural component of all basement membranes. The two subunit genes COL4A1 and COL4A2 are found closely linked in the human and murine genomes and are transcribed divergently from a common promoter. Previously, activating elements had been detected within both genes which are indispensable for efficient transcription. An additional negative regulatory element has now been identified within the third intron of the COL4A2 gene which is able to inhibit transcription of both COL4 genes from their shared promoter, as well as the nonrelated herpes simplex virus thymidine kinase promoter. The element exerts its inhibitory effect largely independently from its relative orientation and distance from the initiation site of transcription. Therefore, the element represents a silencer which is named the "COL4 silencer." The minimal functional silencer could be narrowed down by deletion mapping to a sequence element located within intron 3 of the COL4A2 gene. This motif is specifically recognized by a nuclear protein, named "SILBF," and the binding site of which was determined by footprinting assays. Mutation studies and deletion analysis proved that the presence of this sequence element and its interaction with SILBF is not only essential but also sufficient for the silencing function. We assume that the COL4 silencer plays an important role in the control of overall expression and the balance of divergent transcription of both COL4 genes.

Animals↗
PubMed1995

Purification of the sequence-specific transcription factor CTCBF, involved in the control of human collagen IV genes: subunits with homology to Ku antigen.

The common promoter region of the human collagen type IV genes COL4A1 and COL4A2 comprises a C5TC7 sequence ('CTC box') which is specifically recognized by the recently identified transcription factor CTC box binding factor (CTCBF) involved in the control of divergent transcription of the two genes. This factor has now been purified by affinity chromatography on heparin-agarose and CTC-Sepharose. The CTCBF contains two subunits, CTC75 and CTC85, with molecular weights of 75 and 85 kDa, respectively. Sequence analysis of LysC-derived peptides of the two subunits revealed identity or close homology to p70 and p80 subunits of the human autoantigen Ku. The sequence-specific binding CTCBF represents a presumably tetrameric complex composed of two CTC75/85 heterodimers with an apparent molecular weight of 360-400 kDa. UV crosslinking experiments, the use of Ku-specific antibodies in gel retardation assays and immunoblotting proved that both subunits are involved in sequence-specific interaction with the CTC box motif. The tetrameric complex dissociates in a concentration-dependent manner to CTC75/85 heterodimers which now bind sequence independently to DNA. Three lines of evidence indicate that TATA binding protein (TBP) is additionally involved in the formation of CTCBF: (i) TBP can be detected in purified CTCBF; (ii) the addition of recombinant TBP stimulates formation of the CTCBF-DNA complex; and (iii) antibodies directed against TBP interfere strongly with the formation of the specific protein-DNA complex. The results presented support the idea that the subunits CTC75 and CTC85 (identical or homologous to p70 and p80 of the Ku antigen) are integral parts of CTCBF, and give a first indication of the importance of TBP in the formation of CTCBF.

Amino Acid Sequence↗
PubMed1995

Basement membrane (type IV) collagen.

The collagen IV molecule is 400 nm long and consists of two alpha 1 (IV) and one alpha 2 (IV) chains. Their genes COL4A1 and COL4A2 form a transcription unit in which they are arranged head-to-head and connected by a short common promoter region. Recently, four additional genes have been discovered. They are similarly arranged in the pairs COL4A3-A4 and COL4A5-A6 and code for additional collagen IV isoforms. in the macromolecular network of collagen IV, which provides the scaffold for basement membranes, the molecules are attached and cross-linked via their like end-regions and they interact laterally with their triple-helical domains in a not yet fully understood manner. Collagen IV is involved in interactions with cells and possesses two specific recognition sites for the integrins alpha 1 beta 1 and alpha 2 beta 1.

Amino Acid Sequence↗
PubMed1995

Prevention of glomerular hypertrophy and glomerulosclerosis in Milan normotensive rats by low-protein diet, but not by low-dose captopril treatment.

Milan normotensive rats, which spontaneously develop marked proteinuria (PU) and glomerulosclerosis (GS), were either kept on a normal-protein diet, a normal-protein diet with additional low-dose captopril (CAP), which did not affect blood pressure, or on a low-protein diet. After 8 months PU (79 +/- 25 mg/day) GS (3 +/- 2%) and total glomerular volume (TGV; 27.9 +/- 2.9 mm3/kidney) were significantly lower (p < 0.05) in the low-protein diet group than in both the normal-protein group (PU 583 +/- 210 mg/day, GS 12 +/- 5%, TGV 34.6 +/- 8 mm3/kidney) and the low-CAP group (PU 611 +/- 224 mg/day, GS 16 +/- 6%, TGV 41.8 +/- 8.6 mm3/kidney). In conclusion, the development of glomerular hypertrophy and GS in Milan normotensive rats was reduced by the low-protein diet, but not by low-CAP treatment.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1994

Interaction of enteropathogenic Yersinia enterocolitica with complex basement membranes and the extracellular matrix proteins collagen type IV, laminin-1 and -2, and nidogen/entactin.

The plasmid-encoded virulence factor yersinia adhesin A (YadA) contributes to pathogenicity of Yersinia enterocolitica which might be related to its adhesive potential. Therefore, we have investigated the interaction of Y. enterocolitica with basement membrane (BM) and with the major BM proteins collagen type IV, laminin, and nidogen/entactin. Recombinant YadA-positive but not YadA-negative yersiniae bound specifically to lens capsule BM tissue, as well as purified collagen type IV and the laminin-1 and -2 (formally known as merosin) isoforms. Binding sites are located on the alpha 1 chain of the 58-nm amino-terminal 7sL fragment of collagen type IV and on the elastase-fragment E1 of laminin-1. YadA-mediated binding of yersiniae to collagen type IV was rapid and saturable, it was independent of divalent cations, stable over a wide pH range, and not influenced by higher salt concentrations. D-Glucose and D-galactose did not interfere with binding, indicating a protein-protein interaction. In contrast, adhesion of yersiniae to the laminin-2 isoform occurred also independent of YadA expression and no binding was observed to nidogen/entactin. The results support the hypothesis that adhesion of Y. enterocolitica could contribute to pathogenicity of enteropathogenic yersiniae. Further definition of binding sites for YadA on BM proteins might allow determination of the relevance of Yersinia-BM interactions to infection.

Adhesins, Bacterial↗
PubMed1994

The structural bases of integrin-ligand interactions.

Many extracellular matrix (ECM) proteins, particularly those in the vascular system, use their classical integrin-recognition motif Arg-Gly-Asp (RGD) to interact with integrins. The RGD motif is generally located in flexible peptide loops whose variable conformation enables the relatively few integrins with broad specificity, such as alpha(v)beta(3) and alpha(IIb)beta(3), to bind to a large variety of different ECM proteins. However, certain ECM constituents, such as collagens and laminins, interact with integrins in a conformation-dependent manner, in which both the linear structure and spatial arrangement of the polypeptides are important for the formation of active binding sites. These interactions provide high specificity for the communication of cells with distinct members of the ECM.

Journal Article↗
PubMed1993

Differential effects of DNA-binding proteins on bidirectional transcription from the common promoter region of human collagen type IV genes COL4A1 and COL4A2.

Expression of the heterotrimeric collagen IV (alpha 1(IV))2 alpha 2(IV) is essential for the structural integrity and functional properties of basement membranes. The genes COL4A1 and COL4A2 coding for both subunits are located close to each other on the same chromosome and are transcribed from a common bidirectional promoter element. Binding of at least three different nuclear proteins could be detected within this promoter, a CCAAT-binding protein, Sp1 and a newly identified factor, designated 'CTCBF'. Mutagenesis of binding sites proved that these factors are essential for the efficient transcription of both genes, but revealed differential gene-specific effects. Therefore, the common promoter region of collagen IV does not represent an equally functional bidirectional element, but may be better understood as two overlapping gene-specific promoters with shared elements.

Base Sequence↗
PubMed1993

Interaction of type IV collagen with the isolated integrins alpha 1 beta 1 and alpha 2 beta 1.

The triple-helical cyanogen-bromide-derived fragment CB3[IV] of collagen IV, located 100 nm from the N-terminus of the molecule, contains the binding sites for the integrins alpha 1 beta 1 and alpha 2 beta 1. To investigate the interaction of these integrins and collagen IV, we performed solid-phase and inhibition assays using as receptor isolated alpha 1 beta 1 and alpha 2 beta 1. The ligands used were the binding-site-bearing trimeric peptide CB3[IV] and its shorter tryptic fragments F1-F4. Using titration curves, in which the binding of soluble receptors to coated ligands and the binding of soluble ligands to coated receptors were analyzed, the binding sites for alpha 1 beta 1 and alpha 2 beta 1 were in different but adjacent areas of CB3[IV]. Triple-helical conformation and distinct primary structures were required for the interaction. Dissociation constants (Kd), for the affinity of integrins for collagen IV, were determined in the 1-nM range in the presence of Mn2+ and Mg2+. In the absence of Mn2+, the Kd values indicated a 30-60-fold decrease in the affinities, which for alpha 2 beta 1 was further reduced by adding Ca2+. In the presence of Ca2+ and Mg2+ the affinity of collagen IV for alpha 1 beta 1 was four-times higher than for alpha 2 beta 1.

Amino Acid Sequence↗