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Purpose: Macular Corneal Dystrophy (MCD, MIM #217800) is a category 1 corneal stromal dystrophy as per the current IC3D classification. While characterized by macular stromal deposits, we report a case of MCD type II with isolated bilateral peripheral Decemet membrane opacities, describing the clinical features and results of screening the CHST6 gene and serum sulfated keratan sulfate levels. Observations: A 68-year-old man with an unremarkable past medical and family history presented with bilateral progressive decrease in vision. Ocular exam revealed bilateral clear corneas with the exception of peripheral, round, gray-white discrete deposits at the level of Descemet membrane and decreased central corneal thickness in both eyes. The morphology of the corneal deposits, decreased corneal thickness and the absence of a family history were consistent with MCD, prompting screening of the CHST6 gene. Sanger sequencing followed by allele specific cloning revealed compound heterozygous CHST6 mutations in trans configuration: c.-26C > A, which created a new upstream open reading frame (uORF’), predicted to attenuate translation efficiency of the downstream main ORF; and c.803A > G (p.(Tyr268Cys)), previously associated with MCD. Serum keratan sulfate was reduced but detectable, consistent with the diagnosis of macular corneal dystrophy type II. Conclusions: Although macular corneal dystrophy is classified as a corneal stromal dystrophy with endothelial involvement, we report a case of MCD with dystrophic deposits confined to the peripheral Descemet membrane, indicating that MCD may be associated with isolated endothelial involvement. Keywords: Corneal dystrophy, Macular corneal dystrophy, Corneal endothelial disease

Morquio A syndrome (mucopolysaccharidosis IVA, MPS IVA) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase, resulting in systemic accumulation of the partially degraded glycosaminoglycans (GAGs), keratan sulfate and chondroitin-6-sulfate. The accumulation of these GAGs leads to distinguishing features as skeletal dysplasia with disproportionate dwarfism, short neck, kyphoscoliosis, pectus carinatum, tracheal obstruction, coxa valga, genu valgum, and joint laxity. In the absence of autopsied cases and systemic analysis of multiple tissues, the pathological mechanism of the characteristic skeletal dysplasia associated with the disease largely remains a question. Here we report an autopsied case of a 23-year-old male with MPS IVA, who developed characteristic skeletal abnormalities by 4 months of age and died of severe tracheal obstruction and hypoventilation originating from respiratory muscle weakness from neurological cord deficit due to cord myelopathy at the age of 23. We analyzed postmortem tissues pathohistologically, including the thyroid, lung, lung bronchus, trachea, heart, aorta, liver, spleen, kidney, testes, humerus, knee cartilage, and knee ligament. Examination of the tissues demonstrated systemic storage materials in multiple tissues, as well as severely ballooned and vacuolated chondrocytes in the trachea, humerus, knee cartilage, and lung bronchus. This autopsied case with MPS IVA addresses the importance of tracheal obstruction for morbidity and mortality of the disease, and the pathological findings contribute to a further understanding of the pathogenesis of MPS IVA and the development of novel therapies., Highlights • Severe tracheal obstruction and respiratory failure are the leading causes of morbidity and mortality in MPS IVA patients. • The majority of chondrocytes in the examined locations were enlarged and vacuolated. • Tracheal obstruction is confirmed clinically, pathologically, and radiographically. • Risk factors and surgical intervention of tracheal obstruction should be considered to save the lives of MPS IVA patients. • MPS IVA should be evaluated in a multifaceted approach.

Glycosaminoglycans (GAGs), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS), are the primary biomarkers in patients with mucopolysaccharidoses (MPS); however, little is known about other biomarkers. To explore potential biomarkers and their correlation with GAGs, blood samples were collected from 46 MPS II patients, 34 MPS IVA patients, and 5 MPS IVB patients. We evaluated the levels of 8 pro-inflammatory factors (EGF, IL-1β, IL-6, MIP-1α, TNF-α, MMP-1, MMP-2, and MMP-9), collagen type II, and DS, HS (HS0S, HSNS), and KS (mono-sulfated, di-sulfated) in blood. Eight biomarkers measured were significantly elevated in untreated MPS II patients, compared with those in normal controls: EGF, IL-1β, IL-6, HS0S, HSNS, DS, mono-sulfated KS, and di-sulfated KS. The same eight biomarkers remained elevated in ERT-treated patients. However, only three biomarkers remained elevated in post-HSCT MPS II patients: EGF, mono-sulfated KS, and di-sulfated KS. Post-HSCT patients with MPS II showed that IL-1β and IL-6 were normalized as HS and DS levels decreased. Eight biomarkers were significantly elevated in untreated MPS IVA patients: EGF, IL-1β, IL-6, MIP-1α, MMP-9, HSNS, mono-sulfated KS, and di-sulfated KS, and four biomarkers were elevated in MPS IVA patients under ERT: IL-6, TNF-α, mono-sulfated KS, and di-sulfated KS. There was no reduction of KS in the ERT-treated MPS IVA patient, compared with untreated patients. Two biomarkers were significantly elevated in untreated MPS IVB patients: IL-6 and TNF-α. Reversely, collagen type II level was significantly decreased in untreated and ERT-treated MPS II patients and untreated MPS IVA patients. In conclusion, selected pro-inflammatory factors can be potential biomarkers in patients with MPS II and IV as well as GAGs levels., Highlights • ERT can not normalize DS, HS, and KS levels in MPS II and IVA. • HSCT reduces DS and HS levels more than ERT. • GAG storage in MPS leads to elevation of some cytokines and decrease of collagen type II in blood. • Selected pro-inflammatory factors and collagen type II, as well as GAGs, can be biomarkers in MPS II and MPS IV. • Relation between biomarkers and clinical improvements needs to be addressed with a longitudinal data.

Mucopolysaccharidoses (MPS) are lysosomal storage diseases characterized by deficient degradation of mucopolysaccharides or glycosaminoglycans (GAGs). Abnormal storage of dermatan sulfate, heparan sulfate, keratan sulfate, or hyaluronan occurs in all seven types of MPS due to a deficiency in one of the enzymes in the catabolic pathways of GAGs. MPS disorders are usually multisystemic, with progressive involvement of brain, visceral organs, and bones. However, patients with MPS type IV and VI generally do not have cognitive impairment or central nervous system (CNS) involvement. The characteristic skeletal abnormalities are unique features of MPS IV or Morquio syndrome that are not seen in other types of MPS. Most individuals with MPS appear normal at birth and clinical features develop over time. Each type of MPS has classic or severe presentations as well as attenuated disease forms. Urinary GAG analysis is useful for differential diagnosis and detects total elevations as well as elevated species of GAGs. More recently, liquid chromatography tandem mass spectrometry analysis of GAG-specific disaccharides generated by either enzyme digestion or methanolysis provides a more specific and sensitive solution for screening and monitoring patients with MPS. Enzyme replacement therapy (ERT) has been the mainstay for treating several of the MPSs. Food and Drug Administration approved ERT are available for MPS I, II, IV, and VI. Other therapeutic modalities to target-specific tissues, such as CNS, are being investigated.

The mucopolysaccharidoses are a group of inherited disorders caused by specific enzyme deficiencies in the degradation of the glycosaminoglycans (mucopolysaccharides). Enzyme deficiencies result in the accumulation of glycosaminoglycans in lysosomes of various tissues and in the excessive excretion of partially degraded glycosaminoglycans in urine. Clinical manifestations of the mucopolysaccharidoses depend on the specific enzyme deficiency, the end organ affected, and the accumulation of glycosaminoglycans in the affected organs. In diseases in which the brain is not involved, there is no mental retardation. On the other hand, if the brain is affected and other somatic manifestations are minimal, the coarse features that are characteristic of the mucopolysaccharidoses are not as prominent. Specific degradative lysosomal enzyme deficiencies have been identified for all the mucopolysaccharidoses. The glycosaminoglycans that are stored and excreted in the urine of the various mucopolysaccharidoses are dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin 4/6 sulfates.

Publisher Summary Glycosaminoglycans (GAGs) are a family of highly sulfated, complex, polydisperse linear polysaccharides that display a variety of important biological roles. Based on the difference of repeating disaccharide units comprising GAGs, they can be categorized into four main groups: heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, keratan sulfate, and hyaluronan. This chapter provides the structure analysis of GAGs based on highly efficient isolation/purification techniques, and high-sensitivity, information-rich analytical instruments. GAGs perform a variety of biological functions and play an important role in a number of different diseases. Their activities are mainly triggered by interactions with a wide range of proteins. The structure analysis of GAGs improves the understanding of their biological functions and helps in the development of structure–activity relationships for these important biopolymers. With modern mass spectrometry (MS) techniques, structural information, such as molecular weight, monosaccharide composition, number and position of sulfo groups, composition of disaccharide blocks, and sequence of highly charged sulfated carbohydrates can be obtained. High sensitivity of MS is available for the microanalysis of carbohydrates derived from biological samples. Tandem MS has the potential to become a robust method to completely sequence complex oligosaccharides. Mechanistic studies of oligosaccharide behavior in MS and tandem MS provide the basis for future methods development.

The stroma is a dense connective tissue making up 90% of corneal thickness. The remarkable strength and transparency of the cornea derive from the unique extracellular matrix, elaborated by keratocytes, neural crest-derived cells populating the stroma. Stromal matrix is formed by parallel arrays of collagen fibrils with small uniform diameters and very regular spacing, maintained in association with a unique class of proteoglycans. This organized stromal matrix, along with a specialized class of cellular proteins, crystallins, is required for cornea transparency. A change in the phenotype of the keratocytes during corneal healing leads to deposition of corneal scars and permanent disruption of vision. Understanding the molecular mechanisms of corneal transparency and scarring is essential for devising therapeutic approaches for corneal blindness.

Glycosaminoglycans (GAGs) represent an essential part of glycans in a wide variety of organisms. This chapter will provide an overview of four GAGs in relation to their structure, biosynthesis, and modification; their biological significance; and how these different classes are being used clinically. In general, GAGs are composed of repeating disaccharide units consisting of either N-acetylglucosamine or N-acetylgalactosamine linked to either a hexauronic or a galactose residue. GAGs are a key component in biological functions ranging from anticoagulation and growth pathways to cartilage’s ability to sustain stress during compression. These GAG polysaccharides are able to be synthesized by different tissues to yield unique structures that are able to exhibit functional selectivity. The variety of possible structure sequences and their biological significance is why GAGs remain an important and essential source of drug development studies.

Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hyaluronic acid, and keratan sulfate). Modification of the backbone by sulfation, deacetylation, and epimerization results in unique sequences within GAG molecules, which are instrumental in the binding of a large number of proteins. Investigating the exact roles of GAGs has long been hampered by the lack of appropriate tools, but we have successfully implemented phage display technology to generate a large panel of antibodies against CS, DS, HS, and heparin epitopes. These antibodies provide unique and highly versatile tools to study the topography, structure, and function of specific GAG domains. In this chapter, we describe the selection, characterization, and application of antibodies against specific GAG epitopes.

Proteoglycans have sulfated linear polysaccharide chains, that is, heparan sulfate, heparin, chondroitin sulfates, dermatan sulfate, and keratan sulfate. Many glycosyltransferases and sulfotransferases are involved in biosynthesis of the polysaccharides. Specificities of these enzymes have been mainly determined by evaluating their activities to various acceptor carbohydrates and by analyzing the structure of the products. For the latter purpose, enzymatic hydrolysis using heparitinases, heparinase, and chondroitinases or chemical degradation employing nitrous acid deamination has been effectively used in combination with high‐performance liquid chromatography (HPLC) of the degraded products. As examples, we describe methods for assays and product characterization of sulfotransferases involved in biosynthesis of these polysaccharides, namely heparan sulfate 2‐sulfotransferase, heparan sulfate 6‐sulfotransferases, chondroitin 4‐sulfotransferases, chondroitin 6‐sulfotransferase, N‐acetylgalactosamine 4‐sulfate 6‐sulfotransferase, and N‐acetylglucosamine 6‐sulfotransferases.

Protoglycans are important components of extracellular matrices (ECM) and have multiple functions that depend on both their protein and carbohydrate constituents. They form a special class of glycoproteins with attached long unbranched and highly charged glycosaminoglycan chains. These chains are strongly hydrophilic and dominate the physical properties of protoglycans. But the proteins to which these chains are attached are quite diverse in structure and form several distinct protein families. Protoglycans are most abundant in those tissues where the ECM is highly hydrated. Cartilage and bone are tissues that both contain large expanded ECM, although the composition of these two tissues is strikingly different. In cartilage the major protoglycan is aggrecan, which forms supramolecular aggregates and is important in expanding and hydrating the matrix. It contains mainly chondroitin sulfate and increasing amounts of keratan sulfate with age. There are also lesser amounts of the lower-molecular-weight leucine-rich protoglycans, including decorin, fibromodulin, and lumican, which are collagen fibril-associated, and also biglycan. These also contain chondroitin sulfate, but with varying degrees of epimerization to dermatan sulfate. There is no aggrecan present in bones, and the collagen fibril-associated protoglycans and biglycan are the predominant forms. The cells of bone and cartilage also contain some cell surface integral membrane protoglycans, which predominantly contain heparan sulfate. These are very important in cell–matrix interactions and cell signaling, and control many aspects of chondrocyte and ostoblast function. But as the cell density is low, they do not contribute to a large fraction of the tissue content of protoglycans.

Publisher Summary Glycosaminoglycans (GAGs) include heparin (HP), heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA). These are the polymers of a disaccharide repeat unit, comprising a uronic acid and a hexosamine. Chondroitin and DS arise from a single homogeneous precursor, chondroitin, with the disaccharide repeat unit. KS is the only GAG that does not contain uronic acid; therefore, it is not cleavable by the eliminative enzymatic cleavage. Almost all glucosamines and some of galactose units are 6-sulfated in KS. Proteoglycans are heavily glycosylated proteins, generally consisting of a core protein with one or more covalently linked GAG chains that constitute most of the proteoglycan. The storage function of HS-like GAGs (HSGAGs) is exemplified by the HP found in the granules of mast cells, where these highly sulfated polysaccharides bind to specific granule proteases and store them in an inactive form.

Publisher Summary This chapter reviews current knowledge of the 6-O-HS endosulfatase (Sulf) enzymes, including their unique structural and enzymatic properties as novel extracellular heparan endosulfatases, their regulatory functions in extracellular HS-dependent Wnt and fibroblast growth factors (FGF) signaling in stem cell progenitors, and their roles as well as therapeutic applications in the control of tumor growth and angiogenesis. Conservation of the hydrophilic domain (HD) sequence in association with catalytic and C-terminal domains has enabled identification of Sulf proteins in vertebrates and invertebrates. Sulf enzymes are functionally distinct from other sulfatases, including GlcNR6Sase, in their restricted expression in stem cells, their localization on the cell surface, and their substrate specificity and activity as heparan sulfate (HS)-specific 6-O-endosulfatases. The cell surface localization of Sulf on expressing cells suggests its cell autonomous function. Sulfs were predicted to act on GlcN 6-O-sulfate residues in HS based on their sequence homology with lysosomal GlcNR6Sase, an exoenzyme that removes 6-O-sulfate groups from the nonreducing-terminal GlcN residue of HS, heparin (HP), and keratan sulfate chains.

This chapter describes recent findings and roles of both hyaluronan and its binding proteins in the ocular physiological system, and also discusses them with regard to the pathogenesis of several ocular diseases. Hyaluronan synthase and hyaluronan are expressed in corneal epithelial cells. Hyaluronan stimulates corneal epithelial cell proliferation and migration, while others, such as chondroitin sulfate, keratan sulfate and heparan sulfate, do not increase epithelial migration. Hyaluronan enhances the formation of hemidesmosomes, and has a positive influence on the epithelial resurfacing during the healing phase in corneal wounds, with an increase of corneal hyaluronan occurring in the corneal healing region. Further, the process of corneal epithelial wound healing is modified by fibronectin or matrix metalloprotease (MMP). Versican, a large extracellular matrix proteoglycan, is distributed in corneal epithelium. In normal corneas, the hyaluronan binding protein CD44 is predominantly expressed on the membranes of basal epithelial cells, and its expression is closely correlated with corneal re-epithelialization. Hyaluronan, CD44 and fibronectin collectively play important roles in corneal epithelial wound healing. In recent years, significant advances have been made in understanding the roles of hyaluronan and its binding proteins in the field of visual science. Each ocular tissue physiologically expresses these molecules in a tissue-specific manner, and disordered expressions of these molecules are involved in the pathogenesis of ocular diseases.

Publisher Summary Bone matrix proteoglycans and glycoproteins are proportionally the most abundant constituents of the noncollagenous proteins in bone matrix. Proteoglycan are characterized by the covalent attachment of long chain polysaccharides (glycosaminoglycans, GAGs) to core protein molecules. GAGs are composed of repeating carbohydrate units that are sulfated to varying degrees, and include chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and heparin sulfate (HS). Different subclasses of proteoglycan are generally characterized by the structure of the core protein and by the nature of the GAG. Proteoglycans with protein cores composed of the leucine-rich repeat sequences such as decorin, biglycan, fibromodulin, and osteoadherin, are the predominant form found in mineralized matrix, although hyaluronan-binding forms are present during early stages of osteogenesis. They participate in matrix organization and in regulating growth factor activity. Glycoproteins, such as alkaline phosphatase, osteonectin, RGD-containing proteins (osteoadherin, thrombospondin, fibronectin, vitronectin, osteopontin, bone sialoprotein), fibrillin, and tetranectin are produced at different stages of osteoblastic maturation. They exhibit a broad array of functions ranging from control of cell proliferation, cell-matrix interactions, and mediation of hydroxyapatite deposition. The ectopic expression of bone matrix proteins may also play a significant role in pathological states such as bone metastasis in certain forms of cancer and atherosclerosis.

This chapter focuses on the structure and functions of lumican, which is a small keratan sulfate proteoglycan and whose core protein is homologous to the leucine-rich proteoglycans decorin, biglycan, and fibromodulin. It is expressed in a number of tissues, including cornea (it was originally found in chicken cornea), muscles, aorta, and intestine. It is important in corneal transparency. This protein occurs predominantly as a glycoprotein rather than the highly sulfated proteoglycan present in cornea. It contains 320 amino acids and has a central leucine-rich domain that accounts for 62% of the protein. The chapter presents information on the gene structure, isolation, and the structural and functional sites of lumican.

Aggrecan is a large chondroitin sulfate proteoglycan (CS-PG) that accounts for about 10% of the dry weight of cartilage. It interacts with hyaluronan (HA) via an HA-binding domain. It is usually found as aggrecan, which comprises about 87% chondritin sulfate (CS), 6% keratan sulfate (KS), and 7% protein and has a molecular mass of 2.6 x 106 Da with a core protein of molecular mass approximately of 220 kDa. Aggrecan carries a large number of fixed negatively charged side groups that result in high osmotic pressure in a tissue. The primary role of aggrecan is to swell and hydrate the framework of collagen fibrils in a cartilage. Aggrecan is readily isolated from the cartilage by extraction with sodium acetate and pH. The human aggrecan gene is located on chromosome 15q26. The chapter presents the primary structure and the structural and functional sites in aggrecan.

This chapter focuses on the structure and functions of keratocan, which is one of the three keratan sulfate proteoglycans that are shown to exist in bovine cornea. In cornea, the protein appears to contain one keratan sulfate chain. It is a member of the leucine-rich repeat family of proteins. It contains 11 repeats of the sequence LXXLXLXXNXL/I, where X is any amino acid, and conserved cysteines at the N- and C-terminal ends. The mature protein contains 332 amino acids with a molecular mass of 38,047 Da. There are five potential N -glycosylation sites, a consensus sequence for tyrosine sulfation at the N-terminus. The chapter presents information on the gene structure and the structural and functional sites of the keratocan molecule.

Publisher Summary This chapter presents a study examining location of the keratan sulfate (KS) attachment sites in the hyaluronate binding region (HAABR) of the proteoglycan, aggrecan. HABR from pig laryngeal cartilage was isolated and digested, and the peptides were separated. The KS-containing peptide was isolated by virtue of its high molecular weight relative to other glycosylated and nonglycosylated peptides. It was detected by its positive reaction in ELISA assays using monoclonal antibody 5D4. The peptide was coupled to arylamine-PVDF using (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The membrane was washed with 0.1% aqueous TFA to remove uncoupled material. The membrane was cut into small pieces and placed directly into the standard reaction cartridge, without a glass fiber filter. Tryptic digestion of the aggrecan HABR followed by gel permeation chromatography and reversed-phase HPLC indicated that all the highly sulfated KS in the HABR was in a single tryptic peptide from the C-terminal of the domain. The 27-residue peptide had a high molecular weight (8–15 kDa) as a result of the KS GAG chain(s).

Publisher Summary This chapter defines the incorporation of glucosamine glycosaminoglycans and the influence of corneal epithelium. Multiple extractions of de-epithelialized, minced, or homogenized bovine corneas with 0.15 M NaCl can remove up to 80% of the extractable hexosamine (HexN) without any morphologic effect on either the collagen fibers or lamellar organization. Subsequent extraction of up to 95% of the remaining HexN with 1.0 MCaCl 2 (pH 8) led to disruption of the mature banded-collagen fibers leaving short dense fibrils embedded in a material having the density and fine fibrillar structure of basal lamina. Both extractions produced keratan sulfate (KS) and chondroitin 4-sulfate (C-4-S) in ca. 2:1 ratio and a certain amount of glycoprotein. These experiments are undertaken to investigate the phenomenon and to study the possible influence of the corneal epithelium in the process. The epithelium appears to have a decisive influence on its synthesis, which may be of regulatory character. The destruction of the fibrillary character of the stroma by CaCl 2 does not take place, at least not to the same extent as in de-epithelialyzed swollen corneas. This indicates that this disruption of the fibril structure is not the primary effect of CaCl 2 .

Publisher Summary Proteoglycans are macromolecules that contain a protein core with one or more covalently attached glycosaminoglycan chains. In mammalian tissues, three major classes of glycosaminoglycan are found as components of proteoglycans: chondroitin sulfate/dermatan sulfate, heparan sulfate/heparin, and keratan sulfate. This chapter focuses on more recent methods for isolating and purifying proteoglycans that have proven useful for noncartilage tissues, particularly in cell culture systems. The chapter discusses techniques that were developed for studies of proteoglycans synthesized by rat ovarian granulosa cells in culture. They were designed to solve many of the technical problems associated with noncartilage proteoglycans and are optimized for small-scale cell culture systems. However, they can be scaled up and applied to many other systems including cartilage. There is a discussion on the extraction of proteoglycans. Most isolation procedures used to characterize proteoglycans depend on the physicochemical properties of the glycosaminoglycan chains.

Publisher Summary Propositus is affected by a mucopolysaccharidos different from those already known and possibly caused by the deficiency of an enzyme normally participating in the degradation of keratan and heparan sulfate. In fact, the clinical and radiological features of the propositus represent an association of findings typical of diseases in which heparan sulfate or keratan sulfate accumulate. As acetylglucosamine 6-sulfate is the only functional group common to the repeating units of heparan and keratan sulfate, possibility that the enzyme in the prospositus could be a sulfatase specific for 6-sulfated substrates having the D-glucose configuration has been considered. This hypothesis requires the assumption that at least another sulfatase must exist, specific for 6-sulfated substrates having the D-galactose configuration, whose deificiency might be responsible for the accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio disease. This chapter describes a study to verify these hypotheses in which N-acetyl-glucosamine 6-sulfate, N- acetylgalactosamine 6-sulfate, and galactose 6-sulfate using chlorosulfonic acid, according to the method of Suzuki and Strominger, were synthesized.

Publisher Summary This chapter presents the procedure for purification and assay of endo-β-galactosidase from Eseheriehia freundii. In enzyme isolation The organism from the slant culture is used to inoculate 50-ml flasks, each filled with 10 ml of heart infusion broth containing 0.3% keratan sulfate. The flasks are cultured without shaking at 25 ° for 2 days. Each culture is transferred to a 2-liter flask filled with 1 liter of the same medium and cultured at 25 ° for 2 days. All flasks are plugged with cotton. The cell-free culture medium obtained after centrifugation is used as the source of endo-β-galactosidase. The steps of purification are following: filtration of cell-free culture medium through Amicon H1PIO Hollow Fiber; QAE-Sephadex A-50 chromatography; and CM-Sephadex C-50 Chromatography. Endo-β-galactosidase activity is assayed by using the keratan sulfate isolated from whale nasal cartilage.

Publisher Summary This chapter presents the procedure for purification and assay of endo-β-galactosidase from Flavobacterium keratolyticus. The organism from the slant culture is used to inoculate 50-ml flasks each containing l0 ml of liquid medium. The flasks are incubated without shaking at 25 ° for 2 days. Each culture is then transferred to a 2-liter flask containing 1 liter of liquid medium, and incubated stationary at the same temperature for 5 days. All flasks are plugged with cotton. The cultures are centrifuged at 17,000g for 30 rain. From 15 liters of the liquid culture, 14,060 ml of a clear culture supernatant are obtained. The steps of purification are (1) ammonium sulfate precipitation, (2) sephadex G-100 chromatography, (3) chromatography on a combined column of CM-Sephadex C-50 and DEAE-Sephadex A-50, (4) matrex gel Blue A chromatography, and (5) DEAE-Sephadex A-50 chromatography. The final preparation shows one major band on polyacrylamide gel electrophoresis. Keratan sulfate isolated from whale nasal cartilage is used as substrate in assay.