Ruggero Tenni

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis. A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation. These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth.

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.

Ionic residues influence the stability of collagen triple helices and play a relevant role in the spontaneous aggregation of fibrillar collagens. Collagen types I and II and some of their CNBr peptides were chemically modified in mild conditions with two different protocols. Primary amino groups of Lys and Hyl were N-methylated by formaldehyde in reducing conditions or N-acetylated by sulfosuccinimidyl acetate. The positive charge of amino groups at physiological pH was maintained after the former modification, whereas it was lost after the latter. These chemical derivatizations did not significantly alter the stability of the triple helical conformation of peptide trimeric species. Also the enthalpic change on denaturation was largely unaffected by derivatizations. This implies that no significant variation of weak bonds, either in number or overall strength, and of entropy occur on modification. These properties can probably be explained by the fact that chemically modified groups maintain the ability to form hydrogen bonds.

Acid soluble type I collagen was prepared from foetal and adult bovine tendon and skin and from adult bovine cornea. The degree of hydroxylysine glycosylation and the hydroxylysine di-to monoglycoside ratio as well as the "in vivo" fibril diameters, were shown to be tissue and age-dependent. Fibrils of type I collagens were reconstituted "in vitro" monitoring at 313 nm. The fibrils obtained were examined by electron microscopy. It was shown that the "in vitro" lateral growth of collagen fibrils leads to the formation of fibrils with maximum diameters which may be correlated to those of the corresponding native fibrils. Moreover it is suggested that one of the factors controlling the lateral growth of collagen may be at the level of hydroxylysine glycosylation.

Collagen fibrils were obtained in vitro by aggregation from acid-soluble type I collagen at different initial concentrations and with the addition of decorin core or intact decorin. All specimens were observed by scanning electron microscopy and atomic force microscopy. In line with the findings of other authors, lacking decorin, collagen fibrils undergo an extensive lateral association leading to the formation of a continuous three-dimensional network. The addition of intact decorin or decorin core was equally effective in preventing lateral fusion and restoring the normal fibril appearance. In addition, the fibril diameter was clearly dependent on the initial collagen concentration but not on the presence/absence of proteoglycans. An unusual fibril structure was observed as a result of a very low initial collagen concentration, leading to the formation of huge, irregular superfibrils apparently formed by the lateral coalescence of lesser fibrils, and with a distinctive coil-structured surface. Spots of incomplete fibrillogenesis were occasionally found, where all fibrils appeared made of individual, interwined subfibrils, confirming the presence of a hierarchical association mechanism.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in the SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate leading to cartilage proteoglycan undersulfation. In this work we used the dtd mouse model to study the role of N-acetyl-L-cysteine (NAC), a well known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important prenatal phase of skeletal development and growth we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage proteoglycan sulfation was evaluated by HPLC disaccharide analysis in dtd newborn mice, a marked increase of proteoglycan sulfation was observed in newborns from NAC treated pregnancies compared to the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indi...

Here we summarized what is known at the present about function, structure and effect of mutations in the human prolidase. Among the peptidases, prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement the...

This report describes the biochemical investigations on six patients affected by a moderate form of Osteogenesis Imperfecta (type IV according to the Sillence classification). Biochemical characterization of type I collagen produced by skin fibroblasts showed considerable heterogeneity: in three patients out of six, collagen appeared normal; while in the three others a structural defect in the protein was present. In these probands the mutations were localized in different regions of the triple helix domain (corresponding to peptides alpha 1(I)CB6 and alpha 1(I)CB7). In two probands showing the defect in alpha 1(I)CB7, a decrease of the thermal stability of the protein was present.

In this paper we describe a mild moderate form of osteogenesis imperfecta caused by a point mutation in COL1A1 which converted glycine 85 to valine. The valine substitution introduced into the triple-helical domain of type-I collagen a conformational perturbation causing susceptibility to digestive proteases. In fact, SDS/PAGE of pepsin-treated collagen showed the presence of a faint band, migrating between alpha 1(I) and alpha 2(I), both in the medium and in the cell layer. On trypsin digestion the band, a shortened form of alpha 1(I), had a melting temperature of 39.5 degrees C. If the triple-helical collagen was obtained after trypsin or chymotrypsin digestion of procollagen, two shortened bands were identified; the enzymes cleaved about 40% of the trimers. The mutant procollagen was normally secreted and processed in the extracellular matrix at a normal rate. When native type-I collagen was formed after dextran-sulfate incubation, only chains of normal length were found, suggesting that the fibroblast proteases did not recognize the alteration introduced by the mutation. The effects of glycine 85 to valine substitution are compared with those produced by a previously described arginine substitution of the same residue (Deak et al., 1991).

Prolidase is a Mn(2+)-dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 degrees C was therefore undertaken. For this analysis, an 'on-column' digestion of the N-terminal His tag by Factor Xa was performed. A positive effect of Mn(2+) and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy.

... Antonella SANGALLI ', Antonio ROSS1 ', Monica MOTTES ', Antonella FORLINO ', Ruggero TENNI 3, ... Kuivaniemi, H., Tromp, G. & Prockop, D. J. (1991) Mutations in collagen genes: causes of rare and some common diseases in humans, FASEB J. 5, 2052-2060. ...

The thermal stability of the trimeric species formed by seven type I collagen CNBr peptides was determined at neutral and acidic pH. Melting temperature of peptide trimers and free energy change for monomer to trimer transition were used as indices of trimer stability. A greater stability at neutral pH than at acidic pH was found for all peptides analysed because in most conditions an entropic gain overwhelms an enthalpic cost. Enthalpic reasons are prevailing only in some conditions of the more acidic peptides. The overlap zone of type I collagen fibrils is more basic than the gap zone and is therefore more sensitive to variations of pH from neutral to acidic, e.g. in bone degradation when osteoclasts acidify the lacuna lying between cell and bone. Peptide trimer stability in neutral conditions is influenced also by the chaotropic nature and the concentration of three anions: chloride, sulfate and phosphate. This was more evident for sulfate at the highest concentration used (0.5 M) when a greater stability is caused by entropic reasons. The contribution of hydroxyproline to the stability of peptide trimers is greater at neutral than at acidic pH, particularly at the highest concentration of sulfate. All our data indicate that pH, chaotropic nature and concentration of three anions influence the networks of hydrogen bonds present in the collagen triple helical structure.

As indices of triple helix stability of type I collagen CNBr peptide homotrimers, deltaG degrees for monomer-trimer transitions and melting temperatures were obtained from circular dichroism measurements at increasing temperatures. The data were compared with the stability of the parent native molecule. Peptides were found to have a lower stability than the whole collagen molecule. The general implication is that the coordinated water molecules play a key role in determining collagen triple helical stability and high cooperativity at melting. Other factors (monomer stability, ionic and hydrophobic factors, variations of composition, specific sequences) could also contribute towards peptide stability; these factors could explain the data obtained in the case of peptide alpha1(I) CB3.

This study was undertaken to assess the sensitivity of hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) to monitor bone response to estrogen deficiency and replacement by comparing their excretory patterns in ovariectomized aged (11-14 months old) rats. The ovariectomized (OVX) rats were randomized into two groups: (1) OVX plus vehicle; (2) OVX plus 17 beta-estradiol (17-beta E, 10 micrograms/kg, s.c., 4 days/week). Treatment with 17-beta E started immediately after OVX and continued for 60 days. The collagen catabolites were measured in urine for 1 month before OVX and thereafter for 60 days. In temporal coincidence with urine collection, bone area and bone mineral density (BMD) of lumbar vertebrae, femoral diaphysis and distal metaphysis were measured by dual-energy X-ray absorptiometry. In the untreated rats, BMD of the femoral metaphysis and lumbar vertebrae decreased significantly and the urinary excretion of LP, HP, GHyl and GGHyl increased with different patterns. In the treated rats, 17-beta E replacement prevented the increment in LP excretion, partially prevented the increase in HP excretion, but had no effect on the excretion of GHyl and GGHyl. In conclusion pyridinolines and glycosides have different sensitivities to the bone response to OVX. Glycoside excretion after OVX also reflects metabolic processes not strictly related to bone loss and, in contrast with LP, is not sensitive to estrogen replacement.

Aim of this study was to investigate whether osteoclast activity changes as a consequence of even mild physiological perturbation of plasma calcium as such induced by an oral calcium load. Osteoclast activity was determined indirectly by measuring, in spot urines at two and four hours after oral calcium load, the urinary excretion of hydroxylysylpyridinoline (Pyr), deoxylysylpyridinoline (D-Pyr), hydroxyproline (Hyp) and galactosyl-hydroxylysine (GHyl). The occurrence of the metabolic perturbation of plasma calcium homeostasis was assessed by measuring three indexes: i.e. calcemic response, PTH reduction and calciuric response at times following oral calcium loading. A significant fall of urinary D-Pyr and Pyr followed the perturbation of calcium homeostasis induced by the oral calcium load in two groups of healthy young adult and postmenopausal women. The highest mean percent reduction was observed for D-Pyr and was quantitatively similar in the two groups. Since urinary D-Pyr is the most specific bone resorption marker, it may be inferred that the perturbation of plasma calcium homeostasis induced by an oral calcium load is able to acutely inhibit osteoclast activity. This supports the view that osteoclasts are involved in the short-term error correction of plasma calcium.

Skin, iliac crest cartilage and tendon of a patient affected with Larsen's syndrome were subjected to biochemical and ultrastructural investigation. A substantial increase in the ratio of glucosamine to galactosamine was found both in skin and cartilage. Ultrastructural abnormalities of collagen fibres and proteoglycan filaments were also found in Larsen's tissues. The significance of these findings are discussed.

We have screened type I procollagen synthesized in vitro by skin fibroblasts from several patients with the severe non-lethal form of osteogenesis imperfecta. Cells from one patient synthesized and secreted both normal and a larger amount of abnormal type I procollagen. The abnormal alpha chains are larger in size due to post-translational overmodifications involving the whole triple helical domain. Abnormal collagen heterotrimers had a melting temperature 2.5 degrees-3 degrees C lower than normal ones or from controls. Chemical analysis of collagen in the medium showed a greater degree of both lysyl hydroxylation and hydroxylysyl glycosylation, the major increase in molecular mass of overmodified alpha chains being due to the higher hydroxylysine-bound hexose content. The proband's cells modify proteoglycan metabolism and mineral crystals form in the dermis, possibly a response to abnormal collagen-proteoglycan interactions. These findings can be explained by a small defect in the product of one allele for pro-alpha 1(I) chains: three-quarters of the synthesized type I procollagen molecules are composed of trimers containing one or two chains defective near the C-terminus of the triple helix or in the C-propeptide. The data obtained for this patient confirmed that the severity of clinical manifestations in osteogenesis imperfecta strongly depends on the location and nature of the mutations, and that the phenotype could be a consequence of a collagen defect(s) and its influence on collagen-collagen interactions and collagen interactions with other connective tissue components.

The molecular defect responsible for a case of mild osteogenesis imperfecta (OI) with repeated femoral fractures was investigated. The proband and his mother, who presented minor OI signs but no bone fractures, were shown to produce normal and abnormal type-I procollagen molecules in their dermal fibroblasts. The molecular defect was localized in about half of the proband's pro alpha 1(I) mRNA molecules by chemical cleavage with piperidine of hydroxylamine-reacted mRNA:cDNA heteroduplexes. The corresponding region was reverse-transcribed and amplified by polymerase chain reaction (PCR). Cloning and sequencing of the amplified products revealed in both subjects a G-to-A transition in the first base of codon 901 of the alpha 1(I) triple helical domain, which led to a serine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified genomic DNA from fibroblasts and leukocytes confirmed the heterozygous nature of both patients and proved the absence of mosaicism. The presence of the mutation was excluded in other healthy family members, who were reported to have bluish selerae. The mild phenotypic outcome of this newly characterized mutation contradicts previous findings on glycine substitutions in the C-terminal region of collagen triple helix, most of which caused lethal OI.

Forty-four patients with Osteogenesis Imperfecta (O.I.) were divided into groups on the basis of clinical and genetic criteria and the alterations in collagen and glycosaminoglycans (GAG) in the subjects of each group were examined. The largest group of patients as affected with a mild form of O.I. and showed an increased ratio of type III to type I collagen in skin and an increase of the ratio of hydroxylysine diglycoside to monoglycoside in skin collagen. The group of patients affected with a severe nonlethal form of O.I. appeared to be heterogeneous both from a clinical and from a biochemical point of view. A marked increase of the diglycoside to monoglycoside ratio was observed in skin and urine, whereas the ratio of type III to type I collagen in skin was within the normal range or significantly decreased. Some of these patients also showed alterations involving proteoglycans, e.g. in urinary GAGs a decreased galactosamine to glucosamine ratio could be demonstrated. Similar and more marked alterations involving both collagen and GAG metabolism were observed in five children affected with a lethal form of O.I.

Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2β1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.

Here we summarized what is known at the present about function, structure and effect of mutations in the human prolidase. Among the peptidases, prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement therapy for the prolidase deficiency (PD). Prolidase deficiency is a rare recessive disorder caused by mutations in the prolidase gene and characterized by severe skin lesions. Single amino acid substitutions, exon splicing, deletions and a duplication were described as causative for the disease and are mainly located at highly conserved amino acids in the sequence of prolidase from different species. The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized.