Boris Bätge

Interactions of Fibrillin-1 with Heparin/Heparan Sulfate, Implications for Microfibrillar Assembly

Fibrillin-1 is a major constituent of the 10–12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg45–Thr450) and C-terminal (Asp1528–Arg2731) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp1028–Thr1486) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by β-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.

Fibrillin-1 Interactions with Fibulins Depend on the First Hybrid Domain and Provide an Adaptor Function to Tropoelastin

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.

Changes with age in the urinary excretion of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP).

There is increasing evidence that the measurement of urinary hydroxylysylpyridinoline (HP or PYD) and lysylpyridinoline (LP or DPD) by HPLC (high performance liquid chromatography) is potentially useful in clinical and pharmacological studies. HP and LP are promising markers of bone resorption because their levels in urine reflect the breakdown of mature collagen fibrils mainly of skeletal tissues. HP and LP are two non-reducible cross-links of mature collagen which are formed by a sequence of post-translational modifications. HP is a derivative of three residues of hydroxylysine and is present in almost all mature tissues (e.g. tendon. vessel walls, cartilage, dentine and bone). LP is a derivative of two residues of hydroxylysine and one residue of lysine and is present mainly in dentine and bone. Neither cross-link is found in normal human skin. We have isolated and purified HP and LP from commercially available bone gelatine by a preparative reverse-phase column HPLC. These two components were used as external standards for sample analysis. In the present study we analysed the urinary excretion of HP and LP in a group of 264 male and 279 female healthy subjects aged from 6 months to 65 years. A continuous decline of both cross-link components during childhood paralleled by a decrease of the HP:LP-ratio was observed. The levels of HP and LP were 2.5-5 times higher in infants (0.5-1 year) than in children (5-10 years) and 15-20 times higher than in adults (26-65 years). After the age of 17 years, both parameters remained at low levels. These data allow a precise quantitative monitoring of bone resorption in patients with metabolic bone diseases or during pharmacological interventions.

Comparative study on the thermostability of collagen I of skin and bone : influence of posttranslational hydroxylation of prolyl and lysyl residues

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and afterin vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circulardichroism measurements and limited trypsin digestion. Melting of fibrils from standardizedin vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (Tm) at 43.3°C in 0.05% acetic acid. This is 1.9°C above control skin (Tm=41.4°C), most likely, due to a higher degree of prolyl hydroxylation—0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce theTm slightly (∼1°C). Underhydroxylated bone collagen has aTm which is 2°C below control. Melting temperatures ofin vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3°C, exceeds control bone by 1.4°C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4°C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.

Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts.

Astronauts experiencing long periods of space flight suffer from severe loss of bone tissue, particularly in those bones that carry the body weight under normal gravity. It is assumed that the lack of mechanical load decreases connective tissue biosynthesis in bone-forming cells. To test this assumption, quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity, and hypergravity conditions were investigated by incubating human fibroblast cultures with [3H]-proline for 4, 7, 10, and 20 h during the Spacelab D2-mission in 1993. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being up to 143% higher than in 1 g controls. In contrast, hypergravity samples showed a decrease in collagen synthesis with increasing g, being at the 13% level at 10 g. The relative proportion of collagen in total synthesized protein showed a slight decrease with increasing g. The secretion of collagen by the cells, proline hydroxylation of individual collagen α-chains, and the relative proportions of synthesized collagens I, III, and V were not affected under any of the applied conditions.

Semipreparative isolation of collagen types I, II, III and V by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution.

A simple method for the isolation of alpha-chains of different collagen types was developed. The procedure involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution of separated and defixed collagen alpha-chains. Collagen types I, II, III and V from different porcine tissues were recovered in high quantity (> 95%) and purity (> 98%) as evidenced by amino acid analysis. The procedure can be used for sample quantities smaller than required for conventional methods e.g. chromatographic procedures.

Reduced skin thickness: a new minor diagnostic criterion for the classical and hypermobility types of Ehlers–Danlos syndrome

Background  The diagnosis of Ehlers–Danlos syndrome (EDS) is mainly based on clinical criteria, although in some instances a sound molecular diagnosis is available. Clinical signs can be divided into two categories: one with high diagnostic specificity and the other with low specificity. Despite the fact that reduced skin thickness is one of the dermatological features in patients with EDS, this issue has not been analysed in greater detail.