Lessons from Biomineralisation: the Role of Post-translational Modification

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

Lessons from Biomineralisation: the Role of Post-translational Modification

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

Dermatan-4-O-Sulfotransferase-1 Contributes to the Undifferentiated State of Mouse Embryonic Stem Cells

Mouse embryonic stem cells (mESCs) have the properties of self-renewal and pluripotency. Various signals and growth factors maintain their undifferentiated state and also regulate their differentiation. Glycosaminoglycans are present on the cell surface and in the cell matrix as proteoglycans. Previously, we and other groups reported that the glycosaminoglycan heparan sulfate contributes to both maintenance of undifferentiated state and regulation of mESC differentiation. It has been shown that chondroitin sulfate is needed for pluripotency and differentiation of mESCs, while keratan sulfate is a known marker of human ESCs or induced pluripotent stem cells. We also found that DS promotes neuronal differentiation from mESCs and human neural stem cells; however, the function of DS in the maintenance of mESCs has not yet been revealed. Here, we investigated the role of DS in mESCs by knockdown (KD) or overexpression (O/E) of the dermatan-4-O-sulfotransferase-1 (D4ST1) gene. We found that the activity of the ESC self-renewal marker alkaline phosphatase was reduced in D4ST1 KD mESCs, but, in contrast, increased in D4ST1 O/E mESCs. D4ST1 KD promoted endodermal differentiation, as indicated by an increase in Cdx2 expression. Conversely, Cdx2 expression was decreased by D4ST1 O/E. Wnt signaling, which is also involved in endodermal differentiation, was activated by D4ST1 KD and suppressed by D4ST1 O/E. Collectively, these results demonstrate that D4ST1 contributes to the undifferentiated state of mESCs. Our findings provide new insights into the function of DS in mESCs.

Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.

Plasma Proteomic Analysis in Morquio A Disease

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal disease caused by mutations in the gene encoding the enzymeN-acetylgalactosamine-6-sulfate sulfatase (GALNS), and is characterized by systemic skeletal dysplasia due to excessive storage of keratan sulfate (KS) and chondroitin-6-sulfate in chondrocytes. Although improvements in the activity of daily living and endurance tests have been achieved with enzyme replacement therapy (ERT) with recombinant human GALNS, recovery of bone lesions and bone growth in MPS IVA has not been demonstrated to date. Moreover, no correlation has been described between therapeutic efficacy and urine levels of KS, which accumulates in MPS IVA patients. The objective of this study was to assess the validity of potential biomarkers proposed by other authors and to identify new biomarkers. To identify candidate biomarkers of this disease, we analyzed plasma samples from healthy controls (n=6) and from untreated (n=8) and ERT-treated (n=5, sampled before and after treatment) MPS IVA patients using both qualitative and quantitative proteomics analyses. The qualitative proteomics approach analyzed the proteomic profile of the different study groups. In the quantitative analysis, we identified/quantified 215 proteins after comparing healthy control untreated, ERT-treated MPSIVA patients. We selected a group of proteins that were dysregulated in MPS IVA patients. We identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: fetuin-A, vitronectin, alpha-1antitrypsin, and clusterin. Further studies of cartilage and bone samples from MPS IVA patients will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.

Extracellular Matrix Deposition and Remodeling after Corneal Alkali Burn in Mice

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

Chondroitin Sulfate in USA Dietary Supplements in Comparison to Pharma Grade Products: Analytical Fingerprint and Potential Anti-Inflammatory Effect on Human Osteoartritic Chondrocytes and Synoviocytes

The biological activity of chondroitin sulfate (CS) and glucosamine (GlcN) food supplements (FS), sold in USA against osteoarthritis, might depend on the effective CS and GlcN contents and on the CS structural characteristics. In this paper three USA FS were compared to two pharmaceutical products (Ph). Analyses performed by HPAE-PAD, by HPCE and by SEC-TDA revealed that the CS and GlcN titers were up to −68.8% lower than the contents declared on the labels and that CS of mixed animal origin and variable molecular weights was present together with undesired keratan sulfate. Simulated gastric and intestinal digestions were performed in vitro to evaluate the real CS amount that may reach the gut as biopolymer. Chondrocytes and synoviocytes primary cells derived from human pathological joints were used to assess: cell viability, modulation of the NF-κB, quantification of cartilage oligomeric matrix protein (COMP-2), hyaluronate synthase enzyme (HAS-1), pentraxin (PTX-3) and the secreted IL-6 and IL-8 to assess inflammation. Of the three FS tested only one (US FS1) enhanced chondrocytes viability, while all of them supported synoviocytes growth. Although US FS1 proved to be less effective than Ph as it reduced NF-kB, it could not down-regulate COMP-2; HAS-1 was up-regulated but with a lower efficacy. Inflammatory cytokines were markedly reduced by Ph while a slight decrease was only found for US-FS1.