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U Mayer

Publications and source records attributed to U Mayer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Site-directed mutagenesis and structural interpretation of the nidogen binding site of the laminin gamma1 chain.

A precise molecular map of the nidogen binding site of laminins was obtained by site-directed mutagenesis and structural analysis of the 56 residue LE module gamma1III4 of their gamma1 chain. This demonstrated the crucial importance of the sequence DPNAV (position 800-804) in the disulfide-bonded loop a, with major contributions made by all residues except P801. Different substitutions of these residues emphasized the essential role of the negative charge (D800) and carboxamide group (N802) as well as their spacings and hydrophobic contacts (V804) for interaction, and predict direct contacts of these three residues with a complementary binding region of nidogen. An inactivating A803-V substitution, however, may lead to a distorted loop structure. A lower but still significant contribution originates from the non-contiguous link/loop c sequence LKCIY (positions 815-819) which is spatially close to the loop a sequence. The link residues (L815 and K816) provide main chain hydrogen bonds to N806 and a side chain hydrogen bond to the V804 carbonyl and thus stabilize the conformation of loop a. The side chains of I818 and Y819 together with P842 from loop d form hydrophobic contacts that provide further stability but could possibly also participate in direct ligation. The nidogen binding epitope is therefore localized on a narrow ridge and has a length of approximately 17 angstroms. The data also indicate a strong conservation of the epitope in the laminin gamma1 chains of several invertebrates.

Amino Acid Sequence↗
PubMed1996

Conversion of the Kunitz-type module of collagen VI into a highly active trypsin inhibitor by site-directed mutagenesis.

The recombinant Kunitz protease inhibitor module (domain C5) of human collagen alpha 3(VI) chain was previously shown to lack inhibitory activity for proteases with trypsin-like specificity and some other proteases. We have now prepared mutants in the binding loop region including the P1' site (D2889-->A), the P2' site (F2890-->R) and the P3 site (T2886-->P) and in a more remote region (W2907-->V) either as individual substitutions or combinations of them. These mutants were analyzed for their kinetics of binding to trypsin by surface plasmon resonance and for their capacity to inhibit various proteases. Single substitutions (D-->A, T-->P, W-->V) showed an effect only for D->A which bound to trypsin with Kd = 0.25 microM. A 25-100-fold increase in affinity was observed for the double mutants T-->P/D-->A and F-->R/D-->A and approached the affinity of aprotinin (Kd approximately 0.01 nM) in two different triple mutants. These affinities correlated well with the inhibitory capacities of the mutants for trypsin in the cleavage of a large protein and a small peptide substrate. A similar but not completely identical improvement in inhibitory capacity was also observed for leucocyte elastase but not for thrombin. These data could be interpreted in terms of steric interferences or lack of hydrogen bonding of a few critical residues based on three-dimensional structures available for the C5 domain.

Aprotinin↗
PubMed1996

Molecular analysis of the Arabidopsis pattern formation of gene GNOM: gene structure and intragenic complementation.

The GNOM gene is required for pattern formation along the main body axis of the embryo in the flowering plant Arabidopsis thaliana. Mutations in the GNOM gene alter the asymmetric division of the zygote and interfere with the formation of distinct apical-basal regions in the developing embryo. We have isolated the GNOM gene by positional cloning, characterised its structure and determined the molecular lesions in mutant alleles. Although the predicted 163 kDa GNOM protein has a conserved domain in common with the yeast secretory protein Sec7p, it is most closely related in size and overall similarity to the product of the yeast YEC2 gene, which is not essential for cell viability. Four fully complementing gnom alleles carry missense mutations in conserved regions, seven partially complementing alleles have premature stop codon mutations and two non-complementing alleles have splice-site lesions. Our results suggest that the GNOM protein acts as a complex of identical subunits and that partial complementation may involve low levels of full-length protein generated by inefficient translational read-through.

Alleles↗
PubMed1996

Crystal structure of three consecutive laminin-type epidermal growth factor-like (LE) modules of laminin gamma1 chain harboring the nidogen binding site.

The structure of three consecutive laminin-type EGF-like (LE) modules of mouse laminin gammma1 chain, gamma1III3-5 (positions 738 to 899), has been determined by multiple isomorphous replacement in a crystal of space group p6(4)22 (a=b=74.57 angstroms, c = 185.11 angstroms and gamma = 120 degrees). The crystal structure was refined using restrained crystallographic refinement to an R-factor of 19.72% for 14,983 independent reflections with intensities F(obs)> 0 at 2.1 angstroms resolution, with root mean square deviation of 0.012 angstroms and 1.690 degrees from ideal bond lengths and bond angles, respectively. The final model consisted of 1179 (non-hydrogen) protein atoms within 162 residues and 119 water molecules. The molecule showed a rod-like structure of about 76 angstroms length with individual modules twisted relative to each other by about 70 degrees. Each module has the same disulfide bond connections Cys1-Cys3 (loop a), Cys2-Cys4 (loop b), Cys5-Cys6 (loop c) and Cys7-Cys8 (loop d), the first three being identical to epidermal growth factor (EGF). All three LE modules showed little secondary structure which was mainly restricted to loop d, but they differed in several other details of their structure. The interface contacts between the LE modules are based on hydrogen bonds and hydrophobic interactions between the hydrophobic core of loop d of the preceding module and the first cysteine and an exposed residue in loop b of the following module. Module 4 was previously shown to contribute the major nidogen binding site of laminis and site-directed mutagenesis demonstrated a specific binding role for Asp800, Asn802, Val804 and Tyr819 in loops a and c. The side-chain of these four residues are all located on the surface in a linear array and separated by a distance of 17 angstroms between Tyr819 and Val804. The entire nidogen binding site is stabilized via main-chain hydrogen bonds which are in part derived from the link between loops b and c (residues Leu815 and Lys816). The data demonstrate the unique nature of the LE modules and only a remote similarity to EGF. They also indicate that the crucial residues in the binding loops provide direct contacts with nidogen and explain the synergism between loops a and c which is essential for binding.

Amino Acid Sequence↗
PubMed1996

Structure of the nidogen binding LE module of the laminin gamma1 chain in solution.

The structure of the single LE module between residues 791 and 848 of the laminin gamma1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 phi angles, supplemented by 5 psi and 22 chi1 angles. The main features of the NMR structures are two-stranded antiparallel beta-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long beta-strands that form an antiparallel beta-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen.

Amino Acid Sequence↗
PubMed1996

Structure and multiple conformations of the kunitz-type domain from human type VI collagen alpha3(VI) chain in solution.

BACKGROUND: The Kunitz-type inhibitor motif is found at the C terminus of the human collagen alpha3(VI) chain. This 76-residue module (domain C5) was prepared in recombinant form and showed high stability against proteases; however, it lacked any inhibitory activity against trypsin, thrombin, kallikrein and several other proteases. We have undertaken the determination of the three-dimensional (3D) structure of domain C5 in solution, by nuclear magnetic resonance (NMR), in order to establish the structural basis for the properties of this protein. RESULTS: The 7 N-terminal and 12 C-terminal residues of domain C5 are disordered in the solution structure. The 55-residue core, which shows high homology to bovine pancreatic trypsin inhibitor, retains the characteristic fold of all members of the Kunitz-type inhibitor family. 24 residues of this main structural body show more than one resonance, symptomatic of multiple conformations slowly exchanging on the NMR time scale. In addition, significant proton chemical exchange line broadening is observed for residues in the vicinity of the disulfide bridge between residues 20 and 44: this indicates interconversion, on the micro- to millisecond time scale, between multiple conformations. CONCLUSION: The NMR study demonstrates that domain C5 is a highly dynamic molecule at temperatures studied (between 10 and 30 degrees C). Indeed, some 44% of the main body structure of C5 showed multiple conformations. The existence of multiple conformations was not necessarily expected in view of the conformational constraints imposed by the 3D structure of proteins as rigid as C5; it should therefore be considered in the interpretation of its structural and dynamical properties. The accessibility of the inhibitory binding loop (Gly18 [P4] to Leu25 [P4']) should be relatively unaffected by this conformational exchange and thus would not explain the unusual specificity of C5. Most serine proteinase inhibitors that, like C5, have an arginine at the P1 position inhibit trypsin; the lack of trypsin inhibition of C5 must therefore arise from the amino-acid side-chain composition of the adjoining positions in the binding loop.

Amino Acid Sequence↗
PubMed1996

Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product.

The embryo of the flowering plant Arabidopsis develops by a regular pattern of cell divisions and cell shape changes. Mutations in the KNOLLE (KN) gene affect the rate and plane of cell divisions as well as cell morphology, resulting in mutant seedlings with a disturbed radial organization of tissue layers. At the cellular level, mutant embryos are characterized by incomplete cross walls and enlarged cells with polyploid nuclei. The KN gene was isolated by positional cloning. The predicted KN protein has similarity to syntaxins, a protein family involved in vesicular trafficking. During embryogenesis, KN transcripts are detected in patches of single cells or small cell groups. Our results suggest a function for KN in cytokinesis.

Arabidopsis↗
PubMed1996

Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein.

1. We have used mice with a disrupted mdr 1a P-glycoprotein gene (mdr 1a (-/-) mice) to study the role of P-glycoprotein in the pharmacokinetics of digoxin, a model P-glycoprotein substrate. 2. [3H]-digoxin at a dose of 0.2 mg kg-1 was administered as a single i.v. or oral bolus injection. We focussed on intestinal mucosa and brain endothelial cells, two major pharmacological barriers, as the mdr 1a P-glycoprotein is the only P-glycoprotein normally present in these tissues. 3. Predominant faecal excretion of [3H]-digoxin in wild-type mice shifted towards predominantly urinary excretion in mdr 1a (-/-) mice. 4. After interruption of the biliary excretion into the intestine, we found a substantial excretion of [3H]-digoxin via the gut mucosa in wild-type mice (16% of administered dose over 90 min). This was only 2% in mdr 1a (-/-) mice. Biliary excretion of [3H]-digoxin was not dramatically decreased (24% in wild-type mice versus 16% in mdr 1a (-/-) mice). 5. After a single bolus injection, brain levels of [3H]-digoxin in wild-type mice remained very low, whereas in mdr 1a (-/-) mice these levels continuously increased over a period of 3 days, resulting in a approximately 200 fold higher concentration than in wild-type mice. 6. These data demonstrate the in vivo contribution of intestinal P-glycoprotein to direct elimination of [3H]-digoxin from the systemic circulation and to the pattern of [3H]-digoxin disposition, and they underline the importance of P-glycoprotein for the blood-brain barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Pattern formation in the Arabidopsis embryo: a genetic perspective.

During embryogenesis, a single cell gives rise to different cell types, tissues and organs which are arranged in a biologically meaningful context, or pattern. The resulting basic body organization of higher plants, which is expressed in the seedling, provides a reference system for postembryonic development during which the meristems of the shoot and the root produce the adult body. The seedling may be viewed as the superimposition of two patterns: one along the apical-basal axis of polarity and the other perpendicular to the axis. To analyse mechanisms underlying pattern formation in the embryo, a genetic approach has been taken in Arabidopsis. Mutations in a small number of genes alter one or the other of the two patterns. The mutant phenotypes suggest that early partitioning of the axis is followed by region-specific development, including the formation of the primary shoot and root meristems. The cloning of two genes involved in pattern formation provides a basis for mechanistic studies of how cells adopt specific fates in the developing embryo.

Arabidopsis↗
PubMed1995

Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification.

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase-catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 were identified as major amine acceptor sites. This was confirmed with several mutant proteins, including deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N-terminal sequence (APQQEAL) to an unrelated protein. The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level. For high efficiency amine incorporation an intact domain I is required. The conservation of at least one of the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates.

Amino Acid Sequence↗
PubMed1995

Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif gamma 2III4.

High affinity nidogen binding of laminin-1 (chain composition alpha 1 beta 1 gamma 1) has been previously mapped to a single EGF-like motif gamma 1III4 of its gamma 1 chain. Two more isoforms, laminin-5 (alpha 3 beta 3 gamma 2) and laminin-7 (alpha 3 beta 2 gamma 1), show low and high binding activity, respectively, indicating that the gamma 2 chain is of low affinity. This was confirmed by recombinant production of the homologous EGF-like motif gamma 2III4 of the gamma 2 chain, which has a 100,000-fold lower binding activity than gamma 1III4. The crucial heptapeptide binding sequence Asn-Ile-Asp-Pro-Asn-Ala-Val of gamma 1III4 is modified in gamma 2III4 by replacing both the central Asn and Val by Ser. Changing these replacements to Asn and Val by site-directed mutagenesis enhanced the activity of gamma 2III4 to a level which was only 5-fold lower than that of gamma 1III4. Despite their high sequence identity (77%) motifs gamma 1III4 and gamma 2III4 were also shown to differ considerably in immunological epitopes. This indicates distinctly different functions for laminins which differ in the gamma chain isoform.

Amino Acid Sequence↗
PubMed1995

Binding properties and protease stability of recombinant human nidogen.

Recombinant human nidogen was obtained from transfected kidney cell clones as a 150-kDa protein with a three-globule structure. It was modified by sulfation and O-glycosylation and a lower level of N-glycosylation than mouse nidogen. Recombinant nidogens of both species were, however, indistinguishable in their affinities for laminin-1 and a recombinant laminin gamma 1 chain fragment and showed a similar binding to collagen IV and the heparan sulfate proteoglycan perlecan. The two nidogens were also equivalent in the promotion of ternary complex formation between these ligands, indicating that this function has been conserved during mammalian evolution. Fewer zinc-binding sites could be identified in human nidogen and correlated with a lower capacity of zinc to prevent binding to laminin and collagen IV. Most remarkable was the greater sensitivity of human nidogen to endogenous proteolysis in cell culture, yielding fragments of 90-145 kDa. Studies with several exogenous proteases, including thrombin and leucocyte elastase, showed lack of stability of the N-terminal globular domain G1 in contrast to what was found for mouse nidogen. Since such degradation could be important for basement membrane remodelling, this difference between human and mouse may be biologically significant.

Amino Acid Sequence↗
PubMed1995

[Acute bilateral amaurosis in sphenoid empyema caused by Streptococcus pneumoniae].

PATIENT: A 14-year-old boy suffered from an acute bilateral blindness which occurred in 24-h time, accompanied by headache and raised temperature, with inconspicuous optic nerve head and fundus. After diagnosis of empyema with magnet resonance tomography (MRT) the sphenoid sinus was fenestrated and streptococcus pneumoniae isolated. Liquor and serology being inconspicuous, there was no evidence of leucaemic or autoimmune disease, intoxication or intracranial tumor. CLINICAL COURSE: The condition of the patient improved under systemic antibiotic therapy. The bilateral amaurosis remained and opticus atrophy developed. CONCLUSION: A bilateral amaurosis with descending opticus atrophy as a consequence of a sphenoiditis and spreading inflammation to the meninges and the optic nerve in the area of the chiasm is a rare event. The imaging technique of the MRT offers new opportunities for an early and more pointed diagnosis and therapy.

Adolescent↗
PubMed1995

[Empirical results of adjustment to disability by adolescents with spina bifida and traumatic paraplegia].

Between June 1991 and June 1992, a total of 39 rehabilitees aged 16 to 25 years were examined, i.e., 21 patients suffering from spina bifida and 18 patients suffering from traumatic paraplegia. Diagnostic instruments used were the Freiburg questionnaire on coping with illness (FKV 102), Goldberg et al's questionnaire for the evaluation of subjective wellbeing regarding health, and a self-constructed questionnaire for evaluating satisfaction with rehabilitation. Medical interviews on the present state of health and the present situation were held with each patient. The level of paralysis was taken as an indicator of the motor handicap present. The patients examined showed distinctly less confidence in doctors than adults do. From a subjective point of view, however, the confidence in doctors was good: among the coping strategies chosen, the compliance strategies ranked first and second, respectively, in the two groups. The coping strategy of "cognitive avoidance and dissimilation" is distinctly less common among spina bifida patients than among patients with traumatic paraplegia. They are less inclined to hope for a miracle, and take their handicap much more seriously than adolescents suffering from traumatic paraplegia. The higher the level of paraplegia, the more use is made of the coping strategy of "emotional control and social withdrawal". Regressive tendencies and depressive modes of coping were equally present in both groups. When investigating satisfaction with rehabilitation and with the own person, both groups showed very high satisfaction with the physical independence achieved and the possibilities of acting independently.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Psychological↗
PubMed1994

Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain.

The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.

Amino Acid Sequence↗
PubMed1994

Two non-contiguous regions contribute to nidogen binding to a single EGF-like motif of the laminin gamma 1 chain.

High affinity binding of nidogen to laminin is mediated by an EGF-like repeat gamma 1III4 of the mouse laminin gamma 1 chain and has now been restricted to two short noncontiguous regions of its 56 residue sequence by use of synthetic peptides and recombinant mutants. Disulfide loop a,b of the repeat and a modified loop a,c could completely inhibit binding, with a 5000-fold or 300-fold reduced affinity respectively. Synthetic loops c and d lacked inhibitory activity. Some binding contribution of Tyr819 in loop c was, however, shown by mutation and side chain modification. Together with studies of loop chimeras, this indicated a distinct cooperativity between the two binding sites. The major binding site of loop a was localized to the heptapeptide NIDPNAV (position 798-804). A change of Asp800 to Asn or Ala803 to Val caused a strong reduction in binding activity, while only small effects were observed for the changes Pro801 to Gln and Ile799 to Val. The latter replacement corresponds to the single substitution found in the same region of the Drosophila laminin gamma 1 chain. However, the changes Asn802 to Ser or Val804 to Ser, both known to exist in the laminin gamma 2 chain, were deleterious mutations. This demonstrated conservation of binding structures in laminins of distantly related species, but not between homologous chains of laminin isoforms.

Amino Acid Sequence↗