The CMS19 disease model specifies a pivotal role for collagen XIII in bone homeostasis.

Mutations in the COL13A1 gene result in congenital myasthenic syndrome type 19 (CMS19), a disease of neuromuscular synapses and including various skeletal manifestations, particularly facial dysmorphisms. The phenotypic consequences in Col13a1 null mice (Col13a1-/-) recapitulate the muscle findings of the CMS19 patients. Collagen XIII (ColXIII) is exists as two forms, a transmembrane protein and a soluble molecule. While the Col13a1-/- mice have poorly formed neuromuscular junctions, the prevention of shedding of the ColXIII ectodomain in the Col13a1tm/tm mice results in acetylcholine receptor clusters of increased size and complexity. In view of the bone abnormalities in CMS19, we here studied the tubular and calvarial bone morphology of the Col13a1-/- mice. We discovered several craniofacial malformations, albeit less pronounced ones than in the human disease, and a reduction of cortical bone mass in aged mice. In the Col13a1tm/tm mice, where ColXIII is synthesized but the ectodomain shedding is prevented due to a mutation in a protease recognition sequence, the cortical bone mass decreased as well with age and the cephalometric analyses revealed significant craniofacial abnormalities but no clear phenotypical pattern. To conclude, our data indicates an intrinsic role for ColXIII, particularly the soluble form, in the upkeep of bone with aging and suggests the possibility of previously undiscovered bone pathologies in patients with CMS19.

3D morphometric analysis of calcified cartilage properties using micro-computed tomography.

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (µCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a µ-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA.

In vitro method for 3D morphometry of human articular cartilage chondrons based on micro-computed tomography.

OBJECTIVE: The aims of this study were: to 1) develop a novel sample processing protocol to visualize human articular cartilage (AC) chondrons using micro-computed tomography (µCT), 2) develop and validate an algorithm to quantify the chondron morphology in 3D, and 3) compare the differences in chondron morphology between intact and osteoarthritic AC. METHOD: The developed protocol is based on the dehydration of samples with hexamethyldisilazane (HMDS), followed by imaging with a desktop µCT. Chondron density and depth, as well as volume and sphericity, were calculated in 3D with a custom-made and validated algorithm employing semi-automatic chondron selection and segmentation. The quantitative parameters were analyzed at three AC depth zones (zone 1: 0-10%; zone 2: 10-40%; zone 3: 40-100%) and grouped by the OARSI histological grades (OARSI grades 0-1.0, n = 6; OARSI grades 3.0-3.5, n = 6). RESULTS: After semi-automatic chondron selection and segmentation, 1510 chondrons were approved for 3D morphometric analyses. The chondrons especially in the deeper tissue (zones 2 and 3) were significantly larger (P < 0.001) and less spherical (P < 0.001), respectively, in the OARSI grade 3-3.5 group compared to the OARSI grade 0-1.0 group. No statistically significant difference in chondron density between the OARSI grade groups was observed at different depths. CONCLUSION: We have developed a novel sample processing protocol for chondron imaging in 3D, as well as a high-throughput algorithm to semi-automatically quantify chondron/chondrocyte 3D morphology in AC. Our results also suggest that 3D chondron morphology is affected by the progression of osteoarthritis (OA).

Electromechanical properties of human osteoarthritic and asymptomatic articular cartilage are sensitive and early detectors of degeneration.

OBJECTIVE: To evaluate cross-correlations of ex vivo electromechanical properties with cartilage and subchondral bone plate thickness, as well as their sensitivity and specificity regarding early cartilage degeneration in human tibial plateau. METHOD: Six pairs of tibial plateaus were assessed ex vivo using an electromechanical probe (Arthro-BST) which measures a quantitative parameter (QP) reflecting articular cartilage compression-induced streaming potentials. Cartilage thickness was then measured with an automated thickness mapping technique using Mach-1 multiaxial mechanical tester. Subsequently, a visual assessment was performed by an experienced orthopedic surgeon using the International Cartilage Repair Society (ICRS) grading system. Each tibial plateau was finally evaluated with µCT scanner to determine the subchondral-bone plate thickness over the entire surface. RESULTS: Cross-correlations between assessments decreased with increasing degeneration level. Moreover, electromechanical QP and subchondral-bone plate thickness increased strongly with ICRS grade (ρ = 0.86 and ρ = 0.54 respectively), while cartilage thickness slightly increased (ρ = 0.27). Sensitivity and specificity analysis revealed that the electromechanical QP is the most performant to distinguish between different early degeneration stages, followed by subchondral-bone plate thickness and then cartilage thickness. Lastly, effect sizes of cartilage and subchondral-bone properties were established to evaluate whether cartilage or bone showed the most noticeable changes between normal (ICRS 0) and each early degenerative stage. Thus, the effect sizes of cartilage electromechanical QP were almost twice those of the subchondral-bone plate thickness, indicating greater sensitivity of electromechanical measurements to detect early osteoarthritis. CONCLUSION: The potential of electromechanical properties for the diagnosis of early human cartilage degeneration was highlighted and supported by cartilage thickness and µCT assessments.

Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography.

OBJECTIVE: Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. METHOD: We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland-Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. RESULTS: Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4). CONCLUSIONS: PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

Inhibition of miR-9 de-represses HuR and DICER1 and impairs Hodgkin lymphoma tumour outgrowth in vivo.

MicroRNAs are important regulators of gene expression in normal development and disease. miR-9 is overexpressed in several cancer forms, including brain tumours, hepatocellular carcinomas, breast cancer and Hodgkin lymphoma (HL). Here we demonstrated a relevance for miR-9 in HL pathogenesis and identified two new targets Dicer1 and HuR. HL is characterized by a massive infiltration of immune cells and fibroblasts in the tumour, whereas malignant cells represent only 1% of the tumour mass. These infiltrates provide important survival and growth signals to the tumour cells, and several lines of evidence indicate that they are essential for the persistence of HL. We show that inhibition of miR-9 leads to derepression of DICER and HuR, which in turn results in a decrease in cytokine production by HL cells followed by an impaired ability to attract normal inflammatory cells. Finally, inhibition of miR-9 by a systemically delivered antimiR-9 in a xenograft model of HL increases the protein levels of HuR and DICER1 and results in decreased tumour outgrowth, confirming that miR-9 actively participates in HL pathogenesis and points to miR-9 as a potential therapeutic target.

Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics.

Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA/DNA mixmers, or LNA/RNA mixmers uniquely suited for mimicking RNA structures and for RNA targeting in vitro or in vivo. The focus of this chapter is on LNA antisense, LNA-modified DNAzymes (LNAzymes), LNA-modified small interfering (si)RNA (siLNA), LNA-enhanced expression profiling by real-time RT-PCR and detection and analysis of microRNAs by LNA-modified probes.

LNA-modified oligonucleotides are highly efficient as FISH probes.

Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their thermal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different human-specific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco.

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases.

BACKGROUND: The complex polysaccharide rhamnogalacturonan constitutes a major part of the hairy region of pectin. It can have different types of carbohydrate sidechains attached to the rhamnose residues in the backbone of alternating rhamnose and galacturonic acid residues; the galacturonic acid residues can be methylated or acetylated. Aspergillus aculeatus produces enzymes that are able to perform a synergistic degradation of rhamnogalacturonan. The deacetylation of the backbone by rhamnogalacturonan acetylesterase (RGAE) is an essential prerequisite for the subsequent action of the enzymes that cleave the glycosidic bonds. RESULTS: The structure of RGAE has been determined at 1.55 A resolution. RGAE folds into an alpha/beta/alpha structure. The active site of RGAE is an open cleft containing a serine-histidine-aspartic acid catalytic triad. The position of the three residues relative to the central parallel beta sheet and the lack of the nucleophilic elbow motif found in structures possessing the alpha/beta hydrolase fold show that RGAE does not belong to the alpha/beta hydrolase family. CONCLUSIONS: Structural and sequence comparisons have revealed that, despite very low sequence similarities, RGAE is related to seven other proteins. They are all members of a new hydrolase family, the SGNH-hydrolase family, which includes the carbohydrate esterase family 12 as a distinct subfamily. The SGNH-hydrolase family is characterised by having four conserved blocks of residues, each with one completely conserved residue; serine, glycine, asparagine and histidine, respectively. Each of the four residues plays a role in the catalytic function.

Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.

Crystallization and preliminary X-ray diffraction studies of the heterogeneously glycosylated enzyme rhamnogalacturonan acetylesterase from Aspergillus aculeatus.

Well diffracting crystals of rhamnogalacturonan acetylesterase from Aspergillus aculeatus have been obtained in two polymorphic modifications despite its heterogeneous glycosylation. The best-diffracting crystals (resolution 1.55 A) are orthorhombic. The limit of the diffraction pattern of the other (trigonal) form is 2.5 A. The ability of the enzyme to crystallize appears to depend on the glycosylation of the protein sample. This aspect has been investigated by mass spectrometry, which also showed that the orthorhombic crystals have the same glycosylation as the protein sample used in the crystallization.

The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix.

BACKGROUND: Pectic substances are the major polysaccharide components of the middle lamella and primary cell wall of dicotyledonous plants. They consist of homogalacturonan 'smooth' regions and highly rhamnified 'hairy' regions of rhamnogalacturonan. The backbone in rhamnogalacturonan-l (RG-l), which is composed of alternating galacturonic acid and rhamnose residues, is the substrate for a new class of enzymes known as rhamnogalacturnoases (RGases). RGase A is a novel enzyme implicated in the enzymatic degradation of RG-l. RESULTS: The structure of RGase A from Aspergillus aculeatus has been solved by the single isomorphous replacement method including anomalous scattering (SIRAS method) to 2.0 A resolution. The enzyme folds into a large right-handed parallel beta helix, with a core composed of 13 turns of beta strands. Four parallel beta sheets (PB1, PB1a, PB2 and PB3), formed by the consecutive turns, are typically separated by a residue in the conformation of a left-handed alpha helix. As a consequence of the consecutive turns, 32% of all residues have their sidechains aligned at the surface or in the interior of the parallel beta helix. The aligned residues at the surface are dominated by threonine, aspartic acid and asparagine, whereas valine, leucine and isoleucine are most frequently found in the interior. A very large hydrophobic cavity is found in the interior of the parallel beta helix. The potential active site is a groove, oriented almost perpendicular to the helical axis, containing a cluster of three aspartic acid residues and one glutamic acid residue. The enzyme is highly glycosylated; two N-linked and eighteen O-linked glycosylation sites have been found in the structure. CONCLUSIONS: Rhamnogalacturonase A from A. aculeatus is the first three-dimensional structure of an enzyme hydrolyzing glycoside bonds within the backbone of RG-l. The large groove, which is the potential active site of RGase A, is also seen in the structures of pectate lyases. Two catalytic aspartic acid residues, which have been proposed to have a catalytic role, reside in this area of RGase A. The distance between the aspartic acid residues is consistent with the inverting mechanism of catalysis. The glycan groups bound to RGase A are important to the stability of the crystal, as the carbohydrate moiety is involved in most of the intermolecular hydrogen bonds.

Crystallization and preliminary X-ray studies of rhamnogalacturonase A from Aspergillus aculeatus.

Recombinant rhamnogalacturonase A from Aspergillus aculeatus has been crystallized and X-ray diffraction data has been collected. Crystals were grown by the hanging-drop vapour-diffusion technique, under the conditions 10% PEG 8000, 0.05 M KH(2)PO(4) and 0.1 M sodium acetate buffered at pH 3.5. The crystals diffract beyond 2.0 A resolution and belong to one of the orthorhombic space groups I2(1)2(1)2(1) or I222, with the unit-cell parameters a = 62.9, b = 125.4 and c = 137.0 A. There is one molecule in the asymmetric unit and a solvent content of approximately 54%. The enzyme is highly glycosylated corresponding to 5.9 kDa.

Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani.

Four distinct laccase genes, lcc1, lcc2, lcc3 and lcc4, have been identified in the fungus Rhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. The R. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungus Aspergillus oryzae. Two of the recombinant laccases, r-lcc1 and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66kDa for lcc4 and 50-100kDa for the recombinant lcc1 protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH7, whereas lcc1 has optimal activity at pH6.

Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae.

A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENCI) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENCI amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 degrees C.

Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. Synergism between rhamnogalacturonan degrading enzymes.

A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.