Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model.

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.

Keratan sulfate, an electrosensory neurosentient bioresponsive cell instructive glycosaminoglycan.

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.

Sialylated keratan sulfates on MUC5B are Siglec-8 ligands in the human esophagus.

Human sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed on subsets of immune cells. Siglec-8 is an immune inhibitory Siglec on eosinophils and mast cells, which are effectors in allergic disorders including eosinophilic esophagitis. Inhibition occurs when Siglec-8 is crosslinked by multivalent Siglec ligands in target tissues. Previously we discovered a high-affinity Siglec-8 sialoglycan ligand on human airways composed of terminally sialylated keratan sulfate chains carried on a single protein, DMBT1. Here we extend that approach to another allergic inflammatory target tissue, human esophagus. Lectin overlay histochemistry revealed that Siglec-8 ligands are expressed predominantly by esophageal submucosal glands, and are densely packed in submucosal ducts leading to the lumen. Expression is tissue-specific; esophageal glands express Siglec-8 ligand whereas nearby gastric glands do not. Extraction and resolution by gel electrophoresis revealed a single predominant human esophageal Siglec-8 ligand migrating at >2 MDa. Purification by size exclusion and affinity chromatography, followed by proteomic mass spectrometry, revealed the protein carrier to be MUC5B. Whereas all human esophageal submucosal cells express MUC5B, only a portion convert it to Siglec-8 ligand by adding terminally sialylated keratan sulfate chains. We refer to this as MUC5B S8L. Material from the esophageal lumen of live subjects revealed MUC5B S8L species ranging from ~1-4 MDa. We conclude that MUC5B in the human esophagus is a protein canvas on which Siglec-8 binding sialylated keratan sulfate chains are post-translationally added. These data expand understanding of Siglec-8 ligands and may help us understand their roles in allergic immune regulation.

Expression of low-sulfated keratan sulfate in non-mucinous ovarian carcinoma.

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.

The significant role of glycosaminoglycans in tooth development.

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.

A Biomimetic Synthetic Strategy Can Provide Keratan Sulfate I and II Oligosaccharides with Diverse Fucosylation and Sulfation Patterns.

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.

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

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.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.

Efficient CRISPR/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA.

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.

Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies.

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.

The role of KS GAGs in the microstructure of CXL-treated corneal stroma; a transmission electron microscopy study.

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.

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment.

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.

Structural Characterization and Anticoagulant Activities of a Keratan Sulfate-like Polysaccharide from the Sea Cucumber Holothuria fuscopunctata.

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.

Bone Growth Induction in Mucopolysaccharidosis IVA Mouse.

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.

Mucopolysaccharidosis IVA: Current Disease Models and Drawbacks.

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.

Enhanced Efficiency of the Basal and Induced Apoptosis Process in Mucopolysaccharidosis IVA and IVB Human Fibroblasts.

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.

A novel splicing variant in GALNS in mucopolysaccharidosis IVA and the necessity of re-evaluating primer sequences.

Mucopolysaccharidosis type IVA (MPS IVA; Morquio syndrome type A) is an autosomal recessive disorder caused by defects in the lysosomal hydrolase N-acetylgalactosamine-6-sulfatase (GALNS) gene, leading to progressive systemic skeletal dysplasia. Early diagnosis and early intervention with enzyme replacement therapy are crucial for improving outcomes in these patients. However, a relatively high number of patients are genetically undiagnosed due to high allelic heterogeneity and the absence of robust functional evidence for most variants of the GALNS gene. Herein, we report a novel intronic variant identified with RNA analysis and an allele dropout (ADO) event caused by a common benign variant in the primer-binding site in a Korean boy with MPS IVA. A 28-month-old boy presented with pectus carinatum, kyphoscoliosis, and joint hypermobility with multiple skeletal dysplasia involving the vertebrae and hip joint. Total urinary glycosaminoglycans were elevated with a predominant keratan sulfate fraction, and GALNS (EC 3.1.6.4) activity was significantly decreased in leukocytes. Sanger sequencing was performed; however, only one heterozygous intronic variant with uncertain clinical significance, c.566+3A > T (p.(?)), was identified. As the patient exhibited clinical and biochemical features of MPS IVA, we conducted whole genome sequencing (WGS) of the patient and his family to clarify the molecular diagnosis. WGS revealed a compound heterozygous genotype, c.1019G > A (p.(Gly340Asp)) and c.566+3A > T (p.(?)), in the GALNS gene. On mRNA sequencing, c.566+3A > T, was confirmed to cause exon 5 skipping and a premature stop codon. With subsequent investigation, we discovered that the variant, c.1019G > A, was undetected on initial sequencing because of ADO due to a common benign variant (rs3859024:G > C) at the primer annealing location. We present a novel intronic variant with a splicing defect in the GALNS gene and suggest that clinicians review primer sequences in cases not diagnosed on Sanger sequencing before progressing to diagnostic steps such as WGS.

Findings from the Morquio A Registry Study (MARS) after 6 years: Long-term outcomes of MPS IVA patients treated with elosulfase alfa.

BACKGROUND: The Morquio A Registry Study (MARS) is an ongoing, multinational, observational study of patients with MPS IVA. Key objectives of MARS are to characterize the heterogeneity and natural history of disease and to evaluate long-term effectiveness and safety of elosulfase alfa enzyme replacement therapy (ERT). Enrollment began in September 2014; data on medical history, clinical outcomes, and safety assessments are collected as part of routine care. RESULTS: As of February 2021, 381 subjects from 17 countries had enrolled in MARS: 58 ERT-naïve subjects and 323 ERT-treated subjects (≥1 infusion), with a mean ERT exposure of 5.5 years (SD 2.8) and median age at first ERT treatment of 9.8 years. ERT-treated subjects were younger at diagnosis (median 3.4 vs 6.5 years) relative to ERT-naïve subjects. Among ERT-treated subjects, urinary keratan sulfate (uKS) levels declined from pre-ERT baseline to last follow-up on treatment (mean % change [95% confidence interval]: -52.5% [-57.5%, -47.4%]; n = 115) and 6-min walk test distance remained stable (mean change: -6.1 [-27.6, 15.5] m; n = 131) over a mean follow-up of 5.5 years. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) increased in subjects who were < 18 years of age at ERT initiation (mean change: +0.3 [0.1, 0.4] L and + 0.4 [0.3, 0.5] L; mean follow-up: ∼6 years; n = 82) and were stable in subjects ≥18 years (mean change: 0.0 [-0.0, 0.1] L and 0.0 [-0.1, 0.1] L; mean follow-up: 4.6 years; n = 38). Overall, 148 (47.1%) ERT-treated subjects experienced ≥1 adverse event (AE) and 110 subjects (35%) reported ≥1 serious AE. Drug-related AEs were reported in 39 (12.4%) subjects; the most common were hypersensitivity (9 subjects [2.9%]), urticaria (8 subjects [2.5%]), and pyrexia (7 subjects [2.2%]). CONCLUSIONS: MARS is the longest and largest observational study of MPS IVA patients to date, with a heterogenous population that is representative of the MPS IVA population overall. Data collected over the first 6 years of MARS provide real-world evidence for long-term stabilization of endurance and respiratory function among ERT-treated patients, with no new safety concerns identified.

Membrane-bound mucins of the airway mucosal surfaces are densely decorated with keratan sulfate: revisiting their role in the Lung's innate defense.

Understanding the basic elements of the airway mucosal surfaces and how they form a functional barrier is essential in understanding disease initiation, progression, pathogenesis and ultimately treating chronic lung diseases. Using primary airway epithelial cell cultures, atomic force microscopy (AFM), multiangle light scattering and quartz crystal micro balance with dissipation monitoring techniques, here we report that the membrane bound mucins (MBMs) found in the periciliary layer (PCL) of the airway surface are densely decorated with keratan sulfate (KS). AFM and immunoblotting show that the KS sidechains can be removed enzymatically with keratanase II (KII) treatment, and the antibody accessibility for B2729 (MUC1), MUCH4 (MUC4) and OC125 (MUC16) was substantially enhanced. Light scattering analysis confirmed that KII treatment removed ~40% of the mass from the mucin fractions. Surface binding experiments indicated that MBMs were able to pack into a tighter conformation following KS removal, suggesting that negatively charged KS sidechains play a role in mucin-mucin repulsion and contribute to "space filling" in the PCL. We also observed that soluble filtrate from the common airway pathogen Pseudomonas aeruginosa is capable of stripping KS from MBMs. Altogether, our findings indicate that KS glycosylation of MBMs may play an important role in the integrity of the airway mucosal barrier and its compromise in disease.

A unique subtype of ramified microglia associated with synapses in the rat hippocampus.

To date, a number of studies have reported the heterogeneity of activated microglia. However, there is increasing evidence suggests that ramified, so-called resting, microglia may also be heterogeneous, and they may play diverse roles in normal brain homeostasis. Here, we found that both 5D4 keratan sulfate epitope-positive (5D4+ ) and 5D4-negative (5D4- ) microglia coexisted in the hippocampus of normal rats, while all microglia were negative for the 5D4 epitope in the hippocampus of normal mice. We thus aimed to determine the potential heterogeneity of microglia related to the 5D4 epitope in the normal rat hippocampus. The optical disector analysis showed that the densities of 5D4+ microglia were higher in the stratum oriens of the CA3 region than in other layers and regions. Although both 5D4+ and 5D4- microglia exhibited a ramified morphology, the three-dimensional reconstruction analysis showed that the node numbers, end numbers, and complexity of processes were higher in 5D4+ than in 5D4- microglia. The linear discriminant analysis showed that 5D4+ and 5D4- microglia can be classified into distinct morphometric subtypes. The ratios of contact between synaptic boutons and microglial processes were higher in 5D4+ than in 5D4- microglia. The gene expressions of pro-inflammatory cytokine interleukin-1ß and purinergic receptor P2Y12 (P2Y12 R) were higher in 5D4+ than in 5D4- microglia. Together, these results indicate that at least two different subtypes of ramified microglia coexist in the normal rat hippocampus and also suggest that 5D4+ microglia may represent a unique subtype associated with synapses.