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Applied and Environmental Microbiology Nov 2022The degradation of glycosaminoglycans (GAGs) by intestinal bacteria is critical for their colonization in the human gut and the health of the host. Both colonic and...
The degradation of glycosaminoglycans (GAGs) by intestinal bacteria is critical for their colonization in the human gut and the health of the host. Both colonic and have been reported to degrade GAGs; however, the enzymatic details of the latter remain largely unknown. Our bioinformatic analyses of fecal revealed that their genomes, especially Hungatella hathewayi strains, are an abundant source of putative GAG-specific catabolic enzymes. Subsequently, we isolated a strain, H. hathewayi N2-326, that can catabolize various GAGs. While H. hathewayi N2-326 was as efficient in utilizing chondroitin sulfate A (CSA) and dermatan sulfate as Bacteroides thetaiotaomicron, a well-characterized GAG degrader, it outperformed B. thetaiotaomicron in assimilating hyaluronic acid. Unlike B. thetaiotaomicron, H. hathewayi N2-326 could not utilize heparin. The chondroitin lyase activity of H. hathewayi N2-326 was found to be present predominantly in the culture supernatant. Genome sequence analysis revealed three putative GAG lyases, but only the -chondroitin ABC lyase was upregulated in the presence of CSA. In addition, five CAZyme gene clusters containing GAG metabolism genes were significantly upregulated when grown on CSA. Further characterization of the recombinant chondroitin ABC lyase revealed that it cleaves GAGs predominantly in an exo-mode to produce unsaturated disaccharides as the primary hydrolytic product while exhibiting a higher specific activity than reported chondroitin ABC lyases. -chondroitin ABC lyase represents the first characterized chondroitin lyase from intestinal and offers a viable commercial option for the production of chondroitin, dermatan, and hyaluronan oligosaccharides and also for potential medical applications. An increased understanding of GAG metabolism by intestinal bacteria is critical in identifying the driving factors for the composition, modulation, and homeostasis of the human gut microbiota. In addition, GAG-depolymerizing polysaccharide lyases are highly desired enzymes for the production of GAG oligosaccharides and as therapeutics. At present, the dissection of the enzymatic machinery for GAG degradation is highly skewed toward . In this study, we have isolated an efficient GAG-degrading bacterium from human feces and characterized the first chondroitin ABC lyase from a , which complements the fundamental knowledge of GAG utilization in the human colon. The genomic and transcriptomic analysis of the bacterium shows that might use a distinct approach to catabolize GAGs from that used by . The high specific activity of the characterized chondroitin ABC lyase aids future attempts to develop a commercial chondroitinase for industrial and medicinal applications.
Topics: Humans; Bacteroides; Chondroitin ABC Lyase; Chondroitin Sulfates; Firmicutes; Glycosaminoglycans; Oligosaccharides; Substrate Specificity; Intestines
PubMed: 36342199
DOI: 10.1128/aem.01546-22 -
Frontiers in Immunology 2022Infectious diseases have caused dramatic production decline and economic loss for fish aquaculture. However, the poor understanding of fish disease resistance severely...
Infectious diseases have caused dramatic production decline and economic loss for fish aquaculture. However, the poor understanding of fish disease resistance severely hampered disease prevention. Chinese tongue sole () is an important economic flatfish suffering from vibriosis. Here we used genomic, transcriptomic and experimental approaches to investigate the molecular genetic mechanisms underlying fish vibriosis resistance. A genome-wide comparison revealed that the genes under selective sweeps were enriched for glycosaminoglycan (GAG) chondroitin sulfate (CS)/dermatan sulfate (DS) metabolism. Transcriptomic analyses prioritized synergic gene expression patterns in this pathway, which may lead to an increased CS/DS content in the resistant family. Further experimental evidence showed that carbohydrate sulfotransferases 12 (Chst12), a key enzyme for CS/DS biosynthesis, has a direct antibacterial activity. To the best of our knowledge, this is the first report that the gene has a bactericidal effect. In addition, CS/DS is a major component of the extracellular matrix (ECM) and the selection signatures and fine-tuned gene expressions of ECM-receptor interaction genes indicated a modification in the ECM structure with an enhancement of the barrier function. Furthermore, functional studies conducted on Col6a2, encoding a collagen gene which constitutes the ECM, pointed to that it may act as a cellular receptor for pathogens, thus plays an important role for the invasion. Taken together, these findings provide new insights into the molecular protective mechanism underlying vibriosis resistance in fish, which offers crucial genomic resources for the resistant germplasm breeding and infectious disease control in fish culturing.
Topics: Animals; Transcriptome; Vibrio Infections; Vibrio; Fishes; Chondroitin Sulfates; Genomics
PubMed: 36304468
DOI: 10.3389/fimmu.2022.974604 -
Molecular Genetics and Metabolism... Dec 2022Mucopolysaccharidosis VII (or Sly syndrome) is an autosomal recessive disorder characterised by a deficiency in the enzyme Beta-glucuronidase (). Partial degradation of...
Mucopolysaccharidosis VII (or Sly syndrome) is an autosomal recessive disorder characterised by a deficiency in the enzyme Beta-glucuronidase (). Partial degradation of glycosaminoglycans (GAGs); chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) results in the accumulation of these fragments in the lysosomes of many tissues, eventually leading to multisystem damage. In some cases, early diagnosis on clinical grounds alone can be difficult due to the extreme variability of the clinical presentation and disease progression. We present a case report of a 31-year-old male patient diagnosed with MPS VII at the age of 28, who multiple specialists saw without suspecting the diagnosis due to the unusual presentation. The patient presented with a history of developmental delay, scoliosis, kyphosis, corneal clouding, abnormal gait, short stature, hearing impairment, slightly coarse facial features and progressive deterioration of fine motor skills since childhood. The patient had inguinal hernia repair at around 12 months, bilateral hearing impairment with a left bone-anchored hearing aid, and spinal surgery. During spinal surveillance MPS VII was suspected by a spinal surgeon with interest in MPS, and the diagnosis confirmed with a deficiency in beta-glucuronidase in leucocytes and marginally elevated urinary GAGs. Next-generation sequencing identified two mutations in the gene (OMIM 611499), c.526C > T p.(Leu176Phe) and c.1820G > C p.(Gly607Ala). Although the patient exhibited features of the severe form of non-classical manifestations, his metabolic condition has remained reasonably stable, surviving into adulthood with only symptomatic treatment. We present the ever-expanding phenotypic spectrum of this ultra-rare disease.
PubMed: 36299251
DOI: 10.1016/j.ymgmr.2022.100922 -
ACS Applied Bio Materials Jan 2023Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most...
Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPs─functionalized with positively charged alkaline thiol (TMA)─into adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [ = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.
Topics: Humans; Glycocalyx; Gold; HeLa Cells; Metal Nanoparticles; Glycosaminoglycans; Chondroitin Sulfates
PubMed: 36239448
DOI: 10.1021/acsabm.2c00595 -
Human Gene Therapy Dec 2022Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of...
Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS-deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon-optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV-transduced cells showed supraphysiological levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells, and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS-engrafted animals were restored to 10-20% than that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of MNDU3-IDS LVV-transduced HSPCs for treatment of MPS II.
Topics: Animals; Mice; Humans; Mucopolysaccharidosis II; Leukocytes, Mononuclear; Iduronate Sulfatase; Enzyme Replacement Therapy; Disease Models, Animal; Hematopoietic Stem Cells
PubMed: 36226412
DOI: 10.1089/hum.2022.141 -
Bioengineering & Translational Medicine Sep 2022A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are...
A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are derived from non-human sources along with human cells. Key limitations of such models include a lack of cellular and tissue microenvironment that is representative of human physiology for efficacy studies, as well as the potential for adverse immune responses to animal products for regenerative medicine applications. The use of recombinant extracellular matrix proteins to fabricate tissues can overcome these limitations. We evaluated animal- and non-animal-derived scaffold proteins and glycosaminoglycans for the design of biomaterials for skin reconstruction in vitro. Screening of proteins from the dermal-epidermal junction (collagen IV, laminin 5, and fibronectin) demonstrated that certain protein combinations when used as substrates increase the proliferation and migration of keratinocytes compared to the control (no protein). In the investigation of the effect of components from the dermal layer (collagen types I and III, elastin, hyaluronic acid, and dermatan sulfate), the primary influence on the viability of fibroblasts was attributed to the source of type I collagen (rat tail, human, or bovine) used as scaffold. Furthermore, incorporation of dermatan sulfate in the dermal layer led to a reduction in the contraction of tissues compared to the control where the dermal scaffold was composed primarily of collagen type I. This work highlights the influence of the composition of biomaterials on the development of complex reconstructed skin models that are suitable for clinical translation and in vitro safety assessment.
PubMed: 36176598
DOI: 10.1002/btm2.10297 -
Frontiers in Oncology 2022Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia,...
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of -acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of -acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34 hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.
PubMed: 36158640
DOI: 10.3389/fonc.2022.961473 -
Molecules (Basel, Switzerland) Sep 2022Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where...
Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where they are highly expressed, CS/DS are involved in fundamental processes and various pathologies. The structural diversity of CS/DS domains gave rise to efforts for the development of efficient analytical methods, among which is mass spectrometry (MS), one of the most resourceful techniques for the identification of novel species and their structure elucidation. In this context, we report here on the introduction of a fast, sensitive, and reliable approach based on ion mobility separation (IMS) MS and MS/MS by collision-induced dissociation (CID), for the profiling and structural analysis of CS/DS hexasaccharide domains in human embryonic kidney HEK293 cells decorin (DCN), obtained after CS/DS chain releasing by β-elimination, depolymerization using chondroitin AC I lyase, and fractionation by size-exclusion chromatography. By IMS MS, we were able to find novel CS/DS species, i.e., under- and oversulfated hexasaccharide domains in the released CS/DS chain. In the last stage of analysis, the optimized IMS CID MS/MS provided a series of diagnostic fragment ions crucial for the characterization of the misregulations, which occurred in the sulfation code of the trisulfated-4,5-Δ-GlcAGalNAc[IdoAGalNAc] sequence, due to the unusual sulfation sites.
Topics: Chondroitin Sulfates; Decorin; Dermatan Sulfate; HEK293 Cells; Humans; Lyases; Proteoglycans; Tandem Mass Spectrometry
PubMed: 36144762
DOI: 10.3390/molecules27186026 -
Molecules (Basel, Switzerland) Sep 2022Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western...
Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western Africa is well-known, the recent rise in the number of cases spread through intimate personal contact, particularly in the United States, poses a grave international threat. Previous studies have shown that cell-surface heparan sulfate (HS) is important for vaccinia virus (VACV) infection, particularly the binding of VACV A27, which appears to mediate the binding of virus to cellular HS. Some other glycosaminoglycans (GAGs) also bind to proteins on Orthopoxviruses. In this study, by using surface plasmon resonance, we demonstrated that MPXV A29 protein (a homolog of VACV A27) binds to GAGs including heparin and chondroitin sulfate/dermatan sulfate. The negative charges on GAGs are important for GAG-MPXV A29 interaction. GAG analogs, pentosan polysulfate and mucopolysaccharide polysulfate, show strong inhibition of MPXV A29-heparin interaction. A detailed understanding on the molecular interactions involved in this disease should accelerate the development of therapeutics and drugs for the treatment of MPXV.
Topics: Chondroitin Sulfates; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparitin Sulfate; Monkeypox virus; Pentosan Sulfuric Polyester; Surface Plasmon Resonance; Vaccinia virus
PubMed: 36144634
DOI: 10.3390/molecules27185898 -
JIMD Reports Sep 2022The spondylodysplastic type of Ehlers-Danlos syndrome (spEDS) is caused by genetic defects in the or genes both deranging the biosynthesis of the glycosaminoglycan...
The spondylodysplastic type of Ehlers-Danlos syndrome (spEDS) is caused by genetic defects in the or genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study, we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion-exchange columns, depolymerized with chondroitinase ABC, and analyzed by nLC-MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non-canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, and 59/41. We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.
PubMed: 36101818
DOI: 10.1002/jmd2.12311