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1.
Glycobiology ; 34(5)2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38438145

RESUMO

This review delves into the roles of glycosaminoglycans (GAGs), integral components of proteoglycans, in tooth development. Proteoglycans consist of a core protein linked to GAG chains, comprised of repeating disaccharide units. GAGs are classified into several types, such as hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. Functioning as critical macromolecular components within the dental basement membrane, these GAGs facilitate cell adhesion and aggregation, and play key roles in regulating cell proliferation and differentiation, thereby significantly influencing tooth morphogenesis. Notably, our recent research has identified the hyaluronan-degrading enzyme Transmembrane protein 2 (Tmem2) and we have conducted functional analyses using mouse models. These studies have unveiled the essential role of Tmem2-mediated hyaluronan degradation and its involvement in hyaluronan-mediated cell adhesion during tooth formation. This review provides a comprehensive summary of the current understanding of GAG functions in tooth development, integrating insights from recent research, and discusses future directions in this field.


Assuntos
Glicosaminoglicanos , Ácido Hialurônico , Camundongos , Animais , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Sulfato de Queratano/metabolismo , Sulfatos de Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Odontogênese , Dermatan Sulfato
2.
J Am Chem Soc ; 146(13): 9230-9240, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38494637

RESUMO

Keratan sulfate (KS) is a proteoglycan that is widely expressed in the extracellular matrix of various tissue types, where it performs multiple biological functions. KS is the least understood proteoglycan, which in part is due to a lack of panels of well-defined KS oligosaccharides that are needed for structure-binding studies, as analytical standards, to examine substrate specificities of keratinases, and for drug development. Here, we report a biomimetic approach that makes it possible to install, in a regioselective manner, sulfates and fucosides on oligo-N-acetyllactosamine (LacNAc) chains to provide any structural element of KS by using specific enzyme modules. It is based on the observation that α1,3-fucosides, α2,6-sialosides and C-6 sulfation of galactose (Gal6S) are mutually exclusive and cannot occur on the same LacNAc moiety. As a result, the pattern of sulfation on galactosides can be controlled by installing α1,3-fucosides or α2,6-sialosides to temporarily block certain LacNAc moieties from sulfation by keratan sulfate galactose 6-sulfotransferase (CHST1). The patterns of α1,3-fucosylation and α2,6-sialylation can be controlled by exploiting the mutual exclusivity of these modifications, which in turn controls the sites of sulfation by CHST1. Late-stage treatment with a fucosidase or sialidase to remove blocking fucosides or sialosides provides selectively sulfated KS oligosaccharides. These treatments also unmasked specific galactosides for further modification by CHST1. To showcase the potential of the enzymatic strategy, we have prepared a range of poly-LacNAc derivatives having different patterns of fucosylation and sulfation and several N-glycans decorated by specific arrangements of sulfates.


Assuntos
Galactose , Sulfato de Queratano , Sulfato de Queratano/química , Biomimética , Oligossacarídeos , Carboidrato Sulfotransferases , Proteoglicanas , Galactosídeos , Sulfatos
3.
Molecules ; 29(4)2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38398516

RESUMO

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.


Assuntos
Amino Açúcares , Sulfato de Queratano , Pleura , Animais , Camundongos , Sulfato de Queratano/metabolismo , Pleura/metabolismo , Oligossacarídeos , Polissacarídeos/metabolismo , Epitélio/metabolismo
4.
Glycobiology ; 34(3)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38376199

RESUMO

The roles of keratan sulfate (KS) as a proton detection glycosaminoglycan in neurosensory processes in the central and peripheral nervous systems is reviewed. The functional properties of the KS-proteoglycans aggrecan, phosphacan, podocalyxcin as components of perineuronal nets in neurosensory processes in neuronal plasticity, cognitive learning and memory are also discussed. KS-glycoconjugate neurosensory gels used in electrolocation in elasmobranch fish species and KS substituted mucin like conjugates in some tissue contexts in mammals need to be considered in sensory signalling. Parallels are drawn between KS's roles in elasmobranch fish neurosensory processes and its roles in mammalian electro mechanical transduction of acoustic liquid displacement signals in the cochlea by the tectorial membrane and stereocilia of sensory inner and outer hair cells into neural signals for sound interpretation. The sophisticated structural and functional proteins which maintain the unique high precision physical properties of stereocilia in the detection, transmittance and interpretation of acoustic signals in the hearing process are important. The maintenance of the material properties of stereocilia are essential in sound transmission processes. Specific, emerging roles for low sulfation KS in sensory bioregulation are contrasted with the properties of high charge density KS isoforms. Some speculations are made on how the molecular and electrical properties of KS may be of potential application in futuristic nanoelectronic, memristor technology in advanced ultrafast computing devices with low energy requirements in nanomachines, nanobots or molecular switches which could be potentially useful in artificial synapse development. Application of KS in such innovative areas in bioregulation are eagerly awaited.


Assuntos
Glicosaminoglicanos , Sulfato de Queratano , Animais , Sulfato de Queratano/química , Proteoglicanas/metabolismo , Mamíferos/metabolismo
5.
Glycobiology ; 34(1)2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-37440446

RESUMO

Keratan sulfate glycosaminoglycan is composed of repeating N-acetyllactosamine (LacNAc) disaccharide units consisting of galactose (Gal) and N-acetylglucosamine (GlcNAc), both often 6-O-sulfated. Sulfate contents of keratan sulfate are heterogeneous depending upon the origins. In this study, keratan sulfate is classified as either highly sulfated (in which both GlcNAc and Gal residues are 6-O-sulfated) or low-sulfated (in which only GlcNAc residues are 6-O-sulfated). It is reported that highly sulfated keratan sulfate detected by the 5D4 monoclonal antibody is preferentially expressed in normal epithelial cells lining the female genital tract and in their neoplastic counterparts; however, expression of low-sulfated keratan sulfate in either has not been characterized. In the present study, we generated the 294-1B1 monoclonal antibody, which selectively recognizes low-sulfated keratan sulfate, and performed precise glycan analysis of sulfated glycans expressed on human serous ovarian carcinoma OVCAR-3 cells. We found that OVCAR-3 cells do not express highly sulfated keratan sulfate but rather express low-sulfated form, which was heterogeneous in 294-1B1 reactivity. Comparison of mass spectrometry spectra of sulfated glycans in 294-1B1-positive versus -negative OVCAR-3 cells indicated that the 294-1B1 epitope is likely at least 2, and possibly 3 or more, tandem GlcNAc-6-O-sulfated LacNAc units. Then, using the 294-1B1 antibody, we performed quantitative immunohistochemical analysis of 40 specimens from patients with ovarian cancer, consisting of 10 each of serous, endometrioid, clear cell, and mucinous carcinomas, and found that among them low-sulfated keratan sulfate was widely expressed in all but mucinous ovarian carcinoma.


Assuntos
Adenocarcinoma Mucinoso , Neoplasias Ovarianas , Humanos , Feminino , Sulfato de Queratano/química , Sulfatos , Apoptose , Linhagem Celular Tumoral , Polissacarídeos , Anticorpos Monoclonais
6.
Mar Drugs ; 21(12)2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-38132953

RESUMO

A sulfated polysaccharide (AG) was extracted and isolated from the sea cucumber H. fuscopunctata, consisting of GlcNAc, GalNAc, Gal, Fuc and lacking any uronic acid residues. Importantly, several chemical depolymerization methods were used to elucidate the structure of the AG through a bottom-up strategy. A highly sulfated galactose (oAG-1) and two disaccharides labeled with 2,5-anhydro-D-mannose (oAG-2, oAG-3) were obtained from the deaminative depolymerized product along with the structures of the disaccharide derivatives (oAG-4~oAG-6) identified from the free radical depolymerized product, suggesting that the repeating building blocks in a natural AG should comprise the disaccharide ß-D-GalS-1,4-D-GlcNAc6S. The possible disaccharide side chains (bAG-1) were obtained with mild acid hydrolysis. Thus, a natural AG may consist of a keratan sulfate-like (KS-like) glycosaminoglycan with diverse modifications, including the sulfation types of the Gal residue and the possible disaccharide branches α-D-GalNAc4S6S-1,2-α/ß-L-Fuc3S linked to the KS-like chain. Additionally, the anticoagulant activities of the AG and its depolymerized products (dAG1-9) were evaluated in vitro using normal human plasma. The AG could prolong activated partial thromboplastin time (APTT) in a dose-dependent manner, and the activity potency was positively related to the chain length. The AG and dAG1-dAG3 could prolong thrombin time (TT), while they had little effect on prothrombin time (PT). The results indicate that the AG could inhibit the intrinsic and common coagulation pathways.


Assuntos
Holothuria , Pepinos-do-Mar , Animais , Humanos , Sulfato de Queratano/química , Holothuria/química , Pepinos-do-Mar/química , Polissacarídeos/farmacologia , Polissacarídeos/química , Dissacarídeos , Anticoagulantes/química
7.
Int J Mol Sci ; 24(22)2023 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-38003337

RESUMO

Mucopolysaccharidosis IVA (MPS IVA) is a rare disorder caused by mutations in the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) encoding gene. GALNS leads to the lysosomal degradation of the glycosaminoglyccreasans keratan sulfate and chondroitin 6-sulfate. Impaired GALNS enzymes result in skeletal and non-skeletal complications in patients. For years, the MPS IVA pathogenesis and the assessment of promising drugs have been evaluated using in vitro (primarily fibroblasts) and in vivo (mainly mouse) models. Even though value information has been raised from those studies, these models have several limitations. For instance, chondrocytes have been well recognized as primary cells affected in MPS IVA and responsible for displaying bone development impairment in MPS IVA patients; nonetheless, only a few investigations have used those cells to evaluate basic and applied concepts. Likewise, current animal models are extensively represented by mice lacking GALNS expression; however, it is well known that MPS IVA mice do not recapitulate the skeletal dysplasia observed in humans, making some comparisons difficult. This manuscript reviews the current in vitro and in vivo MPS IVA models and their drawbacks.


Assuntos
Condroitina Sulfatases , Mucopolissacaridose IV , Humanos , Camundongos , Animais , Sulfato de Queratano/metabolismo , Sulfatos de Condroitina , Condrócitos/metabolismo , Modelos Animais de Doenças , Condroitina Sulfatases/genética
8.
Matrix Biol ; 123: 48-58, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37793508

RESUMO

In this review we highlight emerging immune regulatory functions of lumican, keratocan, fibromodulin, biglycan and decorin, which are members of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). These SLRPs have been studied extensively as collagen-fibril regulatory structural components of the skin, cornea, bone and cartilage in homeostasis. However, SLRPs released from a remodeling ECM, or synthesized by activated fibroblasts and immune cells contribute to an ECM-free pool in tissues and circulation, that may have a significant, but poorly understood foot print in inflammation and disease. Their molecular interactions and the signaling networks they influence also require investigations. Here we present studies on the leucine-rich repeat (LRR) motifs of SLRP core proteins, their evolutionary and functional relationships with other LRR pathogen recognition receptors, such as the toll-like receptors (TLRs) to bring some molecular clarity in the immune regulatory functions of SLRPs. We discuss molecular interactions of fragments and intact SLRPs, and how some of these interactions are likely modulated by glycosaminoglycan side chains. We integrate findings on molecular interactions of these SLRPs together with what is known about their presence in circulation and lymph nodes (LN), which are important sites of immune cell regulation. Recent bulk and single cell RNA sequencing studies have identified subsets of stromal reticular cells that express these SLRPs within LNs. An understanding of the cellular source, molecular interactions and signaling consequences will lead to a fundamental understanding of how SLRPs modulate immune responses, and to therapeutic tools based on these SLRPs in the future.


Assuntos
Proteoglicanas de Sulfatos de Condroitina , Proteoglicanos Pequenos Ricos em Leucina , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Decorina/genética , Decorina/metabolismo , Proteoglicanos Pequenos Ricos em Leucina/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Sinais (Psicologia) , Sulfato de Queratano/metabolismo , Biglicano/genética , Biglicano/metabolismo , Matriz Extracelular/metabolismo
9.
Int J Mol Sci ; 24(18)2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37762422

RESUMO

Morquio disease, also called mucopolysaccharidosis IV (MPS IV), belongs to the group of lysosomal storage diseases (LSD). Due to deficiencies in the activities of galactose-6-sulfate sulfatase (in type A) or ß-galactosidase (in type B), arising from mutations in GALNS or GLB1, respectively, keratan sulfate (one of glycosaminoglycans, GAGs) cannot be degraded efficiently and accumulates in lysosomes. This primary defect leads to many cellular dysfunctions which then cause specific disease symptoms. Recent works have indicated that different secondary effects of GAG accumulation might significantly contribute to the pathomechanisms of MPS. Apoptosis is among the cellular processes that were discovered to be affected in MPS cells on the basis of transcriptomic studies and some cell biology experiments. However, Morquio disease is the MPS type which is the least studied in light of apoptosis dysregulation, while RNA-seq analyses suggested considerable changes in the expression of genes involved in apoptosis in MPS IVA and IVB fibroblasts. Here we demonstrate that cytochrome c release from mitochondria is more efficient in MPS IVA and IVB fibroblasts relative to control cells, both under the standard cultivation conditions and after treatment with staurosporine, an apoptosis inducer. This indication of apoptosis stimulation was corroborated by measurements of the levels of caspases 9, 3, 6, and 7, as well as PARP, cleaved at specific sites, in Morquio disease and control fibroblasts. The more detailed analyses of the transcriptomic data revealed which genes related to apoptosis are down- and up-regulated in MPS IVA and IVB fibroblasts. We conclude that apoptosis is stimulated in Morquio disease under both standard cell culture conditions and after induction with staurosporine which may contribute to the pathomechanism of this disorder. Dysregulation of apoptosis in other MPS types is discussed.


Assuntos
Condroitina Sulfatases , Mucopolissacaridose IV , Humanos , Mucopolissacaridose IV/terapia , Estaurosporina/farmacologia , Sulfato de Queratano/metabolismo , Fibroblastos/metabolismo , Apoptose/genética , Condroitina Sulfatases/genética
10.
J Biol Chem ; 299(8): 105052, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37454739

RESUMO

Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is now the third cause of death worldwide, and COVID-19 infection has been reported as an exacerbation factor of them. In this study, we report that the intratracheal administration of the keratan sulfate-based disaccharide L4 mitigates the symptoms of elastase-induced emphysema in a mouse model. To know the molecular mechanisms, we performed a functional analysis of a C-type lectin receptor, langerin, a molecule that binds L4. Using mouse BMDCs (bone marrow-derived dendritic cells) as langerin-expressing cells, we observed the downregulation of IL-6 and TNFa and the upregulation of IL-10 after incubation with L4. We also identified CapG (a macrophage-capping protein) as a possible molecule that binds langerin by immunoprecipitation combined with a mass spectrometry analysis. We identified a portion of the CapG that was localized in the nucleus and binds to the promoter region of IL-6 and the TNFa gene in BMDCs, suggesting that CapG suppresses the gene expression of IL-6 and TNFa as an inhibitory transcriptional factor. To examine the effects of L4 in vivo, we also generated langerin-knockout mice by means of genome editing technology. In an emphysema mouse model, the administration of L4 did not mitigate the symptoms of emphysema as well as the inflammatory state of the lung in the langerin-knockout mice. These data suggest that the anti-inflammatory effect of L4 through the langerin-CapG axis represents a potential therapeutic target for the treatment of emphysema and COPD.


Assuntos
Dissacarídeos , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Animais , Camundongos , Dissacarídeos/farmacologia , Modelos Animais de Doenças , Interleucina-6/genética , Sulfato de Queratano/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/genética , Enfisema Pulmonar/induzido quimicamente , Lectinas Tipo C/metabolismo
11.
Exp Eye Res ; 234: 109570, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37454921

RESUMO

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.


Assuntos
Glicosaminoglicanos , Ceratocone , Suínos , Animais , Glicosaminoglicanos/metabolismo , Sulfato de Queratano/metabolismo , Pepsina A/farmacologia , Pepsina A/metabolismo , Colágeno/metabolismo , Córnea/metabolismo , Substância Própria/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/farmacologia , Raios Ultravioleta , Ceratocone/metabolismo
12.
Ocul Surf ; 29: 521-536, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37355022

RESUMO

Small leucine rich proteoglycans (SLRPs) are the largest family of proteoglycans, with 18 members that are subdivided into five classes. SLRPs are small in size and can be present in tissues as glycosylated and non-glycosylated proteins, and the most studied SLRPs include decorin, biglycan, lumican, keratocan and fibromodulin. SLRPs specifically bind to collagen fibrils, regulating collagen fibrillogenesis and the biomechanical properties of tissues, and are expressed at particularly high levels in fibrous tissues, such as the cornea. However, SLRPs are also very active components of the ECM, interacting with numerous growth factors, cytokines and cell surface receptors. Therefore, SLRPs regulate major cellular processes and have a central role in major fundamental biological processes, such as maintaining corneal homeostasis and transparency and regulating corneal wound healing. Over the years, mutations and/or altered expression of SLRPs have been associated with various corneal diseases, such as congenital stromal corneal dystrophy and cornea plana. Recently, there has been great interest in harnessing the various functions of SLRPs for therapeutic purposes. In this comprehensive review, we describe the structural features and the related functions of SLRPs, and how these affect the therapeutic potential of SLRPs, with special emphasis on the use of SLRPs for treating ocular surface pathologies.


Assuntos
Proteoglicanas de Sulfatos de Condroitina , Proteínas da Matriz Extracelular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Proteoglicanos Pequenos Ricos em Leucina , Decorina , Sulfato de Queratano/metabolismo , Colágeno , Biologia
13.
Ocul Surf ; 29: 388-397, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37327869

RESUMO

Lumican is a keratan sulfate proteoglycan that belongs to the small leucine-rich proteoglycan family. Research has lifted the veil on the versatile roles of lumican in the pathogenesis of eye diseases. Lumican has pivotal roles in the maintenance of physiological tissue homogenesis and is often upregulated in pathological conditions, e.g., fibrosis, scar tissue formation in injured tissues, persistent inflammatory responses and immune anomaly, etc. Herein, we will review literature regarding the role of lumican in pathogenesis of inherited congenital and acquired eye diseases, e.g., cornea dystrophy, cataract, glaucoma and chorioretinal diseases, etc.


Assuntos
Oftalmopatias , Lumicana , Humanos , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Córnea/patologia , Oftalmopatias/metabolismo , Oftalmopatias/patologia , Sulfato de Queratano/fisiologia , Proteoglicanas/fisiologia
14.
Int J Mol Sci ; 24(12)2023 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-37373036

RESUMO

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme, leading to the accumulation of glycosaminoglycans (GAG), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), mainly in cartilage and bone. This lysosomal storage disorder (LSD) is characterized by severe systemic skeletal dysplasia. To this date, none of the treatment options for the MPS IVA patients correct bone pathology. Enzyme replacement therapy with elosulfase alpha provides a limited impact on bone growth and skeletal lesions in MPS IVA patients. To improve bone pathology, we propose a novel gene therapy with a small peptide as a growth-promoting agent for MPS IVA. A small molecule in this peptide family has been found to exert biological actions over the cardiovascular system. This work shows that an AAV vector expressing a C-type natriuretic (CNP) peptide induces bone growth in the MPS IVA mouse model. Histopathological analysis showed the induction of chondrocyte proliferation. CNP peptide also changed the pattern of GAG levels in bone and liver. These results suggest the potential for CNP peptide to be used as a treatment in MPS IVA patients.


Assuntos
Mucopolissacaridose IV , Animais , Camundongos , Sulfato de Queratano , Glicosaminoglicanos , Cartilagem/patologia , Desenvolvimento Ósseo
15.
Exp Eye Res ; 231: 109476, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37068601

RESUMO

The mechanical and physical properties of the cornea originate from the microstructure and composition of its extracellular matrix. It is known that collagen fibrils, with a relatively uniform diameter, are organized in a pseudo-hexagonal array. It has been suggested that proteoglycans and the interaction of their glycosaminoglycan (GAG) side chains with themselves and collagen fibrils are important for collagen fibril organization inside the cornea. There are several diseases such as keratoconus in which the regular collagen fibrillar packing becomes distorted causing corneal optical and mechanical properties to be compromised. The primary purpose of the present work was to investigate the role of GAGs on the microstructure of corneal extracellular matrix before and after corneal crosslinking (CXL) treatment. For this purpose, keratan sulphates (KS) were removed from corneal samples using the keratanase enzyme and the CXL procedure was used to crosslink the specimens. The transmission electron microscopy was then used to characterize the diameter of collagen fibrils and their interfibrillar spacing. It was found that KS GAG depletion increased the collagen interfibrillar spacing while the CXL treatment significantly decreased the interfibrillar spacing. The enzyme and CXL treatments had an insignificant effect on the diameter of collagen fibrils. The underlying mechanisms responsible for these observations were discussed in terms of the assumption that GAG chains form duplexes that behave as tiny ropes holding collagen fibrils in place.


Assuntos
Córnea , Substância Própria , Colágeno , Microscopia Eletrônica de Transmissão , Matriz Extracelular , Sulfato de Queratano , Glicosaminoglicanos
16.
PLoS One ; 18(4): e0284292, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37053259

RESUMO

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.


Assuntos
Anormalidades Musculoesqueléticas , Osteocondrodisplasias , Animais , Bovinos , Camundongos , Transporte Biológico , Sulfato de Queratano , Mamíferos , Nucleotídeos , Osteocondrodisplasias/genética , Difosfato de Uridina
17.
Gene Ther ; 30(1-2): 107-114, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-35581402

RESUMO

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.


Assuntos
Condroitina Sulfatases , Mucopolissacaridose IV , Humanos , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/terapia , Sistemas CRISPR-Cas , Edição de Genes , Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Condroitina Sulfatases/uso terapêutico , Sulfato de Queratano/metabolismo , Sulfato de Queratano/uso terapêutico , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo
18.
Glycobiology ; 33(2): 150-164, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36373215

RESUMO

This report describes the isolation and characterization of two new antibodies, R-6C (IgM) and R-13E (IgM), which were generated in C57BL/6 mice (Mus musculus) using the Tic (JCRB1331) human induced pluripotent cell (hiPSC) line as an antigen, and their comparisons with two existing antibodies, R-10G (IgG1) and R-17F (IgG1). Their epitopes were studied by western blotting after various glycosidase digestions, binding analyses using enzyme-linked immunosorbent assays (ELISAs) and microarrays with various synthetic oligosaccharides. The minimum epitope structures identified were: Siaα2-3Galß1-3GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)ß1 (R-6C), Fucα1-2Galß1-3GlcNAcß1-3Galß1 (R-13E), Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)ß1 (R-10G), and Fucα1-2Galß1-3GlcNAß1-3Galß1-4Glc (lacto-N-fucopentaose I) (R-17F). Most glycoprotein epitopes are expressed as O-glycans. The common feature of these epitopes is the presence of an N-acetyllactosamine type 1 structure (Galß1-3GlcNAc) at their nonreducing termini, followed by a type 2 structure (Galß1-4GlcNAc); this arrangement comprises a type 1-type 2 motif. This motif is also shared by TRA-1-60, a traditional onco-fetal antigen. In contrast, the R-10G epitope has a type 2-type 2 motif. Among these antibodies, R-17F and R-13E exhibit cytotoxic activity toward hiPSCs. R-17F and R-13E exhibit extremely high similarity in the amino acid sequences in their complementarity-determining regions (CDRs), which is consistent with their highly similar glycan recognition. These antibodies are excellent tools for investigating the biological functions of glycoconjugates in hiPSCs/hESCs; they could be useful for the selection, isolation and selective killing of such undifferentiated pluripotent stem cells.


Assuntos
Sulfato de Queratano , Oligossacarídeos , Camundongos , Animais , Humanos , Sulfato de Queratano/química , Camundongos Endogâmicos C57BL , Oligossacarídeos/química , Polissacarídeos/química , Epitopos/química , Imunoglobulina G , Imunoglobulina M
19.
Adv Neurobiol ; 29: 117-162, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255674

RESUMO

Proteoglycans, and especially their GAG components, participate in numerous biologically significant interactions with growth factors, chemokines, morphogens, guidance molecules, survival factors, and other extracellular and cell-surface components. These interactions are often critical to the basic developmental processes of cellular proliferation and differentiation, as well as to both the onset of disease sequelae and prevention of disease progression. In many tissues, proteoglycans and especially their glycosaminoglycan (GAG) components are mediators of these processes. The GAG family is characterized by covalently linked repeating disaccharides forming long unbranched polysaccharide chains. Thus far in higher eukaryotes, the family consists of chondroitin sulfate (CS), heparin/heparan sulfate (HS), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronan (HA). All GAG chains (except HA) are characteristically modified by varying amounts of esterified sulfate. One or more GAG chains are usually found in nature bound to polypeptide backbones in the form of proteoglycans; HA is the exception. In the nervous system, GAG/proteoglycan-mediated interactions participate in proliferation and synaptogenesis, neural plasticity, and regeneration. This review focuses on the structure, chemistry and function of GAGs in nervous system development, disease, function and injury response.


Assuntos
Sulfatos de Condroitina , Glicosaminoglicanos , Humanos , Glicosaminoglicanos/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato , Sulfato de Queratano , Ácido Hialurônico , Heparitina Sulfato/metabolismo , Proteoglicanas , Heparina , Dissacarídeos , Sulfatos/metabolismo , Sistema Nervoso
20.
Pathol Oncol Res ; 28: 1610537, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36277959

RESUMO

Objective: To reduce the risk of locoregional recurrence, the addition of neoadjuvant concurrent chemoradiotherapy (CCRT) is recommended before surgical management for rectal cancer patients. However, despite identical tumor histology, individual patient response to neoadjuvant CCRT varies greatly. Accordingly, a comprehensive molecular characterization that is used to predict CCRT efficacy is instantly needed. Methods: Pearson's chi-squared test was utilized to correlate dehydrogenase/reductase 9 (DHRS9) expression with clinicopathological features. Survival curves were created applying the Kaplan-Meier method, and the log-rank test was conducted to compare prognostic utility between high and low DHRS9 expression groups. Multivariate Cox proportional hazards regression analysis was applied to identify independent prognostic biomarkers based on variables with prognostic utility at the univariate level. Results: Utilizing a public transcriptome dataset, we identified that the DHRS9 gene is the most considerably upregulated gene related to epithelial cell differentiation (GO: 0030855) among rectal cancer patients with CCRT resistance. Employing immunohistochemical staining, we also demonstrated that high DHRS9 immunoexpression is considerably associated with an aggressive clinical course and CCRT resistance in our rectal cancer cohort. Among all variables with prognostic utility at the univariate level, only high DHRS9 immunoexpression was independently unfavorably prognostic of all three endpoints (all p ≤ 0.048) in the multivariate analysis. In addition, applying bioinformatic analysis, we also linked DHRS9 with unrevealed functions, such as keratan sulfate and mucin synthesis which may be implicated in CCRT resistance. Conclusion: Altogether, DHRS9 expression may serve as a helpful predictive and prognostic biomarker and assist decision-making for rectal cancer patients who underwent neoadjuvant CCRT.


Assuntos
Sulfato de Queratano , Neoplasias Retais , Humanos , Sulfato de Queratano/uso terapêutico , Imuno-Histoquímica , Intervalo Livre de Doença , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Retais/terapia , Quimiorradioterapia , Terapia Neoadjuvante , Prognóstico , Mucinas/uso terapêutico , Oxirredutases/uso terapêutico , Estudos Retrospectivos
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