Targeted delivery of nitric oxide via a 'bump-and-hole'-based enzyme-prodrug pair.

The spatiotemporal generation of nitric oxide (NO), a versatile endogenous messenger, is precisely controlled. Despite its therapeutic potential for a wide range of diseases, NO-based therapies are limited clinically due to a lack of effective strategies for precisely delivering NO to a specific site. In the present study, we developed a novel NO delivery system via modification of an enzyme-prodrug pair of galactosidase-galactosyl-NONOate using a 'bump-and-hole' strategy. Precise delivery to targeted tissues was clearly demonstrated by an in vivo near-infrared imaging assay. The therapeutic potential was evaluated in both rat hindlimb ischemia and mouse acute kidney injury models. Targeted delivery of NO clearly enhanced its therapeutic efficacy in tissue repair and function recovery and abolished side effects due to the systemic release of NO. The developed protocol holds broad applicability in the targeted delivery of important gaseous signaling molecules and offers a potent tool for the investigation of relevant molecular mechanisms.

Detection of GM1-gangliosidosis in newborn dried blood spots by enzyme activity and biomarker assays using tandem mass spectrometry.

GM1-gangliosidosis is a rare autosomal recessive lysosomal storage disease caused by deficiency of ß-galactosidase (GLB1). Newborn screening (NBS) may be warranted in the near future given the initiation of a number of gene therapy clinical trials. Here, we report a tandem mass spectrometry (MS/MS) enzymatic assay of GLB1 using dried blood spots (DBS), and the demonstration that GLB1 activities in newborn DBS from seven GM1-gangliosidosis patients are well below those measured in random newborn DBS. MS/MS analysis of two glycan biomarkers, dp5 and A2G2, shows high elevation in newborn DBS from GM1-gangliosidosis compared to the levels in the nonaffected reference range.

Bottom-up single-molecule strategy for understanding subunit function of tetrameric ß-galactosidase.

In this paper, we report an example of the engineered expression of tetrameric ß-galactosidase (ß-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric ß-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10-4 per base). In addition, we studied the kinetics of the mistranslated ß-gal at the single-molecule level.

Structural and functional analysis of tomato ß-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme.

Plant ß-galactosidases hydrolyze cell wall ß-(1,4)-galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato ß-galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of ß-(1,4)-galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1-7 that belongs to the ß-galactosidase/exo-ß-(1,4)-galactanase subfamily. The enzyme can hydrolyze a wide range of plant-derived (1,4)- or 4-linked polysaccharides, and shows a strong ability to attack ß-(1,4)-galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with ß-d-galactose. TBG4 comprises a catalytic TIM barrel domain followed by three ß-sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 ß-galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards ß-(1,6)-galactobiose, and ~0.6-fold activity towards ß-(1,4)-galactobiose, compared with wild-type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant ß-galactosidases towards ß-1,4 and ß-1,6 linkages.

Inhibition of oxidative stress in rostral ventrolateral medulla improves impaired baroreflex sensitivity in stroke-prone spontaneously hypertensive rats.

Reactive oxygen species (ROS) in rostral ventrolateral medulla (RVLM) of brainstem contribute to sympathoexcitation and are critically involved in the pathogenesis of hypertension. Baroreflex sensitivity (BRS) is a valuable prognostic parameter of the autonomic nervous system, and is impaired in hypertension. The aim of the present study was to determine whether or not a chronic reduction of ROS in the RVLM improves impaired BRS in hypertensive rats. We transfected adenovirus vectors encoding either manganese superoxide dismutase (AdMnSOD) or ß-galactosidase (AdLacZ) into the RVLM of stroke-prone spontaneously hypertensive rats (SHRSP). We measured BRS using the spontaneous sequence method. BRS was significantly lower in SHRSPs than in Wistar-Kyoto rats. In the AdMnSOD-transfected SHRSP, blood pressure, heart rate, and sympathetic nervous system activation were significantly decreased from day 5 after the gene transfer. BRS in the AdMnSOD-transfected SHRSP was significantly increased from day 4 after the gene transfer with the reduction of ROS in the RVLM. Furthermore, in the AdMnSOD-transfected SHRSP, intravenous infusion of atropine dramatically decreased BRS. In contrast, in the AdLacZ-transfected SHRSP, atropine did not decrease BRS. These results suggest that chronic reduction of ROS in the local RVLM improves the impaired BRS in SHRSP through inhibition of the sympathetic component.

AFos inhibits phenylephrine-mediated contractile dysfunction by altering phospholamban phosphorylation.

Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing beta-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (ß-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore ß-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human ß-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased ß-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

Studies on cell adhesion and recognition. I. Extent and specificity of cell adhesion triggered by carbohydrate-reactive proteins (glycosidases and lectins) and by fibronectin.

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.

Binding of elastin peptides to S-Gal protects the heart against ischemia/reperfusion injury by triggering the RISK pathway.

Elastin peptides (EPs) generated by hydrolysis of elastic fibers by elastinolytic enzymes display a wide spectrum of biological activities. Here, we investigated their influence on rat heart ischemia-mediated injury using the Langendorff ex vivo model. EPs, i.e., kappa elastin, at 1.32- and 660-nM concentrations, when administered before the ischemia period, elicited a beneficial influence against ischemia by accelerating the recovery rate of heart contractile parameters and by decreasing significantly creatine kinase release and heart necrosis area when measured at the onset of the reperfusion. All effects were S-Gal-dependent, as being reproduced by (VGVAPG)3 and as being inhibited by receptor antagonists, such as lactose and V14 peptide (VVGSPSAQDEASPL). EPs interaction with S-Gal triggered NO release and activation of PI3-kinase/Akt and ERK1/2 in human coronary endothelial cells (HCAECs) and rat neonatal cardiomyocytes (RCs). This signaling pathway, as designated as RISK, for reperfusion injury salvage kinase pathway, was shown to be responsible for the beneficial influence of EPs on ischemia/reperfusion injury on the basis of its inhibition by specific pharmacological inhibitors. EPs survival activity was attained at a concentration averaging that present into the blood circulation, supporting the contention that these matrikines might offer a natural protection against cardiac injury in young and adult individuals. Such protective effect might be lost with aging, since we found that hearts from 24-month-old rats did not respond to EPs.

Functional characterization of persimmon ß-galactosidase gene DkGAL1 in tomato reveals cell wall modification related to fruit ripening and radicle elongation.

Cell wall metabolism during fruit ripening is a highly organized process that involves complex interplay among various cell wall hydrolases. Among these cell wall hydrolases, ß-galactosidase has been identified to participate in cell wall metabolism via its ability to catalyze galactosyl metabolism from the large and complex side chains of cell walls. In this study, the galactose content in the pericarp increased during persimmon fruit ripening, but cell wall galactosyl residues decreased, indicating a relationship between galactose metabolism and persimmon fruit ripening. Expression of a previously isolated ß-galactosidase gene, DkGAL1, increased 25.01-fold during fruit ripening. Heterologous expression of DkGAL1 under the CaMV 35S promoter in tomato accelerated on-plant and postharvest fruits ripening. The fruit firmness of one of transgenic line, OE-18, was 23.83% lower than that of WT at the breaker stage. The transgenic fruits produced more ethylene by promoting the expression of ethylene synthesis-related genes and cell wall degradation-related genes. Overexpression of DkGAL1 in tomato also reduced cell-to-cell adhesion and promoted both wider intercellular spaces and less cell compaction in transgenic fruit structures. Moreover, DkGAL1 was involved in seed germination and radicle elongation in transgenic tomato seeds. These results confirm the role of DkGAL1 in fruit ripening and suggest that this gene alters galactose metabolism in the fruit, which can promote ripening and reduce cellular adhesion. In addition, the role of DkGAL1 is not limited to fruit softening; DkGAL1 was also involved in seed germination and radicle elongation in transgenic tomato seeds.

Diagnostic challenge for the rare lysosomal storage disease: Late infantile GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare lysosomal storage disorder caused by GLB1 mutations. Because of its extreme rarity and symptoms that overlap with other neurodegenerative diseases, its diagnosis is sometimes challenging, especially in the late infantile form with less severe phenotype. We aim to expand the clinical and genetic spectrum of late infantile GM1 gangliosidosis. METHODS: We confirmed a diagnosis of GM1 gangliosidosis based on GLB1 mutations and/or the deficiency of ß-galactosidase activity. We identified the first two cases by whole-exome sequencing, and then the other six cases by direct sequencing of GLB1 with enzyme analysis. RESULTS: All eight patients presented with developmental delay or regression during late infancy and later developed epilepsy, mostly intractable generalized tonic seizures. No clinical signs of storage disorders were noted except for skeletal abnormalities. Interestingly, we found aspartate transaminase (AST) elevations alone with normal alanine transaminase (ALT) levels in all patients. The recurrent mutation, p.D448V in GLB1, accounted for 50.0% of total alleles in our cohort. CONCLUSIONS: With a high index of clinical suspicion, skeletal survey and AST level would be important for early diagnosis of GM1 gangliosidosis. In addition, we would highlight the clinical usefulness of whole-exome sequencing in the diagnosis of non-classical presentation of ultra-rare neurodegenerative disease in children.

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35.

Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.

Lysosomal multienzyme complex: biochemistry, genetics, and molecular pathophysiology.

Lysosomal enzymes sialidase (alpha-neuraminidase), beta-galactosidase, and N-acetylaminogalacto-6-sulfate sulfatase are involved in the catabolism of glycolipids, glycoproteins, and oligosaccharides. Their functional activity in the cell depends on their association in a multienzyme complex with lysosomal carboxypeptidase, cathepsin A. We review the data suggesting that the integrity of the complex plays a crucial role at different stages of biogenesis of lysosomal enzymes, including intracellular sorting and proteolytic processing of their precursors. The complex plays a protective role for all components, extending their half-life in the lysosome from several hours to several days; and for sialidase, the association with cathepsin A is also necessary for the expression of enzymatic activity. The disintegration of the complex due to genetic mutations in its components results in their functional deficiency and causes severe metabolic disorders: sialidosis (mutations in sialidase), GM1-gangliosidosis and Morquio disease type B (mutations in beta-galactosidase), galactosialidosis (mutations in cathepsin A), and Morquio disease type A (mutations in N-acetylaminogalacto-6-sulfate sulfatase). The genetic, biochemical, and direct structural studies described here clarify the molecular pathogenic mechanisms of these disorders and suggest new diagnostic tools.

The activity of three glycosidases (ß-Ν-acetyloglucosaminidase, α-mannosidase, and ß-galactosidase) in the follicular fluid and in the maturation medium affects bovine oocyte maturation.

We studied the role of follicular fluid's (FF) glycosidase (α-mannosidase [α-ΜΑΝ], ß-Ν-acetyloglucosaminidase [NAGASE], ß-galactosidase [ß-GAL]) activity during IVM of bovine oocytes. Oocytes were allocated into two groups according to the follicular size (small follicle [SF]: 2-5 mm, large follicle [LF]: >5-8 mm). In experiment 1, cumulus-oocyte complexes (COCs) quality was evaluated according to morphologic criteria (grades A, B-C, D); oocyte (n = 801) nuclear maturation was assessed after 24 hours of incubation. Bovine embryos were produced in vitro in groups (experiment 2, n = 1503 oocytes) or individually (experiment 3, n = 50 oocytes). More grade-A and -BC COCs were collected from SF and LF groups, respectively (P < 0.05). Maturation rate (experiment 1) and cleavage rate (experiments 2 and 3) were similar in SF and LF groups. Activity of all glycosidases in FF was higher (P < 0.05) in SF group than in LF group, whereas in maturation medium of SF group it was, overall, significantly lower than in that of LF (experiments 2 and 3). In FF of SF group, NAGASE positively associated with grade-A oocytes and negatively with BC oocytes; increased ß-GAL was associated with degenerated oocytes. Cleavage rate in LF group, related negatively to NAGASE and positively to α-MAN in maturation medium. These results indicate that during maturation, COCs release NAGASE and consume ß-GAL, but differences probably exist between individual and group maturation.

Regional localization of Fyn in adult brain; studies with mice in which fyn gene was replaced by lacZ.

Mutant mice in which beta-galactosidase gene (lacZ) was inserted into fyn locus were generated by homologous recombination in embryonic stem cells to examine the Fyn expression in the central nervous system. In adult brain, intensive beta-galactosidase activity was observed in olfactory bulb, cerebellum and hippocampus of the limbic system; the subcellular distribution of the activity was apparent not only in cell body but also in neural processes, and homozygous mutant mice live-born displayed an anatomical abnormality in the neural cell layer of the hippocampal formation. In spinal cord it was specifically expressed in dorsal horn, and in brain stem it was more characteristic in the sensory pathway, suggesting roles of Fyn in the sensory nervous network. In the white matter area, it was intense at postnatal day 10 but not detectable in adult, suggesting Fyn's role in myelinization.

Role of beta-galactosidase and elastin binding protein in lysosomal and nonlysosomal complexes of patients with GM1-gangliosidosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of beta-galactosidase (GLB1). The GLB1 gene gives rise to the GLB1 lysosomal enzyme and to the elastin binding protein (EBP), involved in elastic fiber deposition. GLB1 forms a complex with protective protein cathepsin A (PPCA), alpha neuraminidase (NEU1), and galactosamine 6-sulphate sulfatase (GALNS) inside lysosomes, while EBP binds to PPCA and NEU1 on the cell surface. We investigated the function of the GLB1 and EBP mutated proteins by analyzing the clinical, genetic, and cellular data of 11 G(M1)-gangliosidosis patients. Their molecular analysis, followed by expression studies, lead to the identification of four new and 10 known GLB1 mutations. Some common amino acid substitutions [c.1445G>A (p.Arg482H), c.622C>T (p.Arg208His), c.175C>T (p.Arg59Cys) and c.176G>A (p.Arg59His)] were present in the GLB1 enzyme of several patients, all of Mediterranean origin, suggesting a common origin. Western blotting analyses against GLB1, EBP, and PPCA proteins showed that the identified mutations affect GLB1 enzyme activity and/or stability. The c.1445G>A (p.Arg482His), c.175C>T (p.Arg59Cys), c.733+2T>C, c.1736G>A (p.Gly579Asp), and c.1051C>T (p.Arg351X) GLB1 mutations, affect the stabilization of PPCA probably because they hamper the interaction between GLB1/EBP and PPCA within the multiprotein complex. The amount of EBP was normal, but the detection of impaired elastogenesis in such patients suggests an alteration in its function. We conclude that the presence of genetic lesions in both GLB1 and EBP coding region does not directly predict impaired elastogenesis and that elastic fiber assembly has to be evaluated specifically in each case. Nevertheless, the degree of EBP involvement may be linked to specific clinical findings.

Expression in bacteria of beta-galactosidase fusion proteins carrying antigenic determinants of the two X gene products of bovine leukemia virus.

A cDNA corresponding to the bovine leukemia virus post-envelope region (X gene) was subcloned into the lambda gt11 expression vector. Two large protein fragments corresponding respectively to the long and short open reading frames of the X region were expressed as beta-galactosidase fusion proteins. These products were specifically recognized by sera from bovine leukemia virus-infected cattle.

Roles of beta-galactosidase of B lymphocytes and sialidase of T lymphocytes in inflammation-primed activation of macrophages.

The outer surface of mouse B lymphocytes carries constitutive and inducible beta-galactosidase isozymes. A brief (30 min) treatment of B lymphocytes with lysophosphatidylcholine (lyso-Pc) immediately induced an approximate 3-fold higher beta-galactosidase activity than the constitutive isozyme of untreated B lymphocytes. Thus, the lyso-Pc-inducible isozyme is not a de novo enzyme. Outer surface of mouse T lymphocytes carries constitutive (non-Neu-1) and inducible (Neu-1) sialidase isozymes. The lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes were required for conversion of vitamin D3-binding protein (Gc protein) to a potent macrophage activating factor. This enzymatic generation of the macrophage activating factor was mediated via enzyme-associated receptors.

Analysis of orthologous hrcA genes in Escherichia coli and Bacillus subtilis.

The hrcA gene codes for a transcriptional repressor protein interacting with the CIRCE operator thereby reducing expression of the groE operon of more than 120 bacterial species. At least in Bacillus subtilis, the activity of the HrcA protein is modulated by the GroE chaperonin system. We amplified the hrcA gene from five different bacterial species and analyzed its activity in Escherichia coli and Bacillus subtilis. While those from Clostridium acetobutylicum and Staphylococcus aureus turned out to be active, those of Helicobacter pylori, Lactococcus lactis and Thermotoga maritima were inactive in E. coli, but that of T. maritima turned out to repress expression of the reporter gene in B. subtilis. All these results strongly suggest to us a specific recognition of HrcA by the GroE chaperonin system.

beta-Galactosidase and its significance in ripening mango fruit.

The fruit extracts of ripening cv. Harumanis mango contained a number of glycosidases and glycanases. Among the glycosidases, beta-D-galactosidase (EC 3.2.1.23) appeared to be the most significant. The enzyme activity increased in parallel with increase in tissue softness during ripening. Mango beta-galactosidase was fractionated into three isoforms, viz. beta-galactosidase I, II and III by a combination of chromatographic procedures on DEAE-Sepharose CL-6B, CM-Sepharose and Sephacryl S-200 columns. Apparent Km values for the respective beta-galactosidase isoforms for p-nitrophenyl beta-D-galactoside were 3.7, 3.3 and 2.7 mM, and their Vmax values were 209, 1024 and 62 nkat mg-1 protein. Optimum activity occurred at ca pH 3.2 for beta-galactosidase I and II, and pH 3.6 for beta-galactosidase III. Mango beta-galactosidase and its isoforms have galactanase activity, and the activity of the latter in the crude extracts generally increased during ripening. The close correlation between changes in beta-galactosidase activity, tissue softness, and increased pectin solubility and degradation suggests that beta-galactosidase might play an important role in cell wall pectin modification and softening of mango fruit during ripening.