International Journal of Biological Macromolecules 72 (2015) 282–289

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Review

Keratan sulfate: An up-to-date review Vitor H. Pomin ∗ Program of Glycobiology, Institute of Medical Biochemistry Leopoldo de Meis, and University Hospital Clementino Fraga Filho, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-913, Brazil

a r t i c l e

i n f o

Article history: Received 2 July 2014 Received in revised form 20 August 2014 Accepted 23 August 2014 Available online 29 August 2014 Keywords: Carbohydrate-based drug Cartilage Eye drop Glycosaminoglycan Keratan sulfate Inflammation

a b s t r a c t Keratan sulfate (KS) is a glycosaminoglycan (GAG) type consisted of a sulfated poly-N-acetyl lactosamine chain. Besides acting as a constitutive molecule of the extracellular matrices, this GAG also plays a role as a hydrating and signaling agent in cornea and cartilage tissues. Inasmuch, KS is widely explored in the pharmaceutical industry. This review will cover the major achievements described in the literature of 2010–2014 concerning this GAG. Discussion about KS’ roles in physiopathological conditions, as target or therapeutic molecule in diseases, methods of analysis and detection as well as KS-related enzymes, metabolism and developmental biology is properly provided. © 2014 Elsevier B.V. All rights reserved.

Contents 1.

2.

3.

4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Scope of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role in physiopathological conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Carcinoma of female genital tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Neural regeneration and plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Mucopolysaccharidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . As a therapeutic or target molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. As suppressor of cartilage damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. As regulators of inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. In malignant cellular apoptotic process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. As suppressors of amyotrophic lateral sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of detection and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KS-related enzymes, synthesis and developmental biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Correspondence to: R. Prof. Rodolpho Paulo Rocco, 255, HUCFF 4A01, Ilha do Fundão, Rio de Janeiro, RJ 21941-913, Brazil. Tel.: +55 21 3938 2939; fax: +55 21 3938 2090. E-mail addresses: [email protected], [email protected] http://dx.doi.org/10.1016/j.ijbiomac.2014.08.029 0141-8130/© 2014 Elsevier B.V. All rights reserved.

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OH OR1

Gal H

(β1→4)

H O

H

(β1→3) H

GlcNAc

H OR2 O

H

O

O OH

HO

H

H H

H3COC

O NH H

n

R1,2 = H orSO 3 -; R1 < R2 = SO3Fig. 1. Structural representation of keratan sulfate. It is a polymer composed of disaccharide repetitions of alternating 3-linked ␤-galactopyranose (Gal) and 4-linked N-acetylglucosamine (GlcNAc). Sulfation occurs at 6-position of any monosaccharide but more often at the GlcNAc.

1. Introduction 1.1. Structure Keratan sulfate (KS) is a glycosaminoglycan (GAG) type widely found in the extracellular matrices (ECM) of certain tissues, such as cornea, cartilages and bone. KS is composed of a sulfated poly-Nacetyl lactosamine backbone. Its structure is formed by alternating 3-linked ␤-galactose (Gal) and 4-linked N-acetyl-␤-glucosamine (GlcNAc) units displayed in disaccharide repeating building blocks within a polysaccharide chain [1,2]. Although both units can be 6-O-sulfated, this modification occurs more often at the GlcNAc units [3] (Fig. 1). KS is the only GAG type which does not bear an acidic residue [1–3] such as glucuronic acid commonly seen in chondroitin sulfates, dermatan sulfate, hyaluronic acid, and heparin/heparan sulfate [4]; or iduronic acid commonly seen in dermatan sulfate and heparin/heparan sulfate [4]. Instead of these acidic units, KS has the neutral sugar Gal [1–4], and this characteristic gives to KS a less acidic potential in solution, once sulfation is the only acidic component of its structure. KS chains are generally found structurally attached to a protein core forming thus proteoglycans (PGs) [5]. KS chains can be either N-linked to asparagine residues (named as KS I) or O-linked to serine or threonine residues (named as KS II) [6]. While KS I occurs more often at the corneal tissue, KS II happens more frequently at the cartilages [1]. Both KS I and II possess a mixture of non-sulfated (Gal-GlcNAc), mono-sulfated (Gal-GlcNAc6S), and disulfated (Gal6S-GlcNAc6S) disaccharide units within their chains [6]. The keratan sulfate PGs (KSPGs) can be either primarily composed of KS chains like the family of small leucine-rich ECM PGs such as keratocan, mimecan, lumican, fibromodulin, osteomodulin, and osteoadherin, or just containing few KS chains, as the least abundant GAG type, like aggrecan, which is largely composed of chondroitin sulfate [1,5,6]. 1.2. Function In terms of biological actions, KS is a functional component of PGs from cornea, cartilage and bone tissues. In cornea, the high abundance of KSPGs is related in maintaining the proper hydration levels of this tissue. This is relevant in order to keep constant the transparency of the tissue [1,7]. This factor is extremely important to allow the light beams passing through and converging precisely at the retina in order to generate the right visual effect [1,7]. Anyone could assume unexpected if undesirable structural changes on KS chains of the corneal PGs led to visual dysfunctions. And that is exactly what happens in certain visual disorders like macular corneal dystrophy and keratoconus [1,6,7]. While the former is characterized by defections or changes on the patterns of sulfation, the latter is occasioned by malfunctioning in KS chain formation.

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Nonetheless, both disorders are occasioned by distortions in fibrils (collagens and PGs) organization in the cornea resulting increased opacity of the tissue [1,6,7]. The use of KS as an active ingredient in eye drops may help to restore the healthier condition. This explains why KS is widely explored in the market as an active ingredient of eye drops. Like in cornea, in cartilages KS is also well-known to play a role in keeping balanced the hydration properties of the tissue, especially as a component of aggrecan which is considerably efficient in confer resistance to physical stress and loads on the tissue. Aggrecan is considered the molecular assembly of the highest molecular weight of the body. In bones, KS seems to play a primary role as structural component of certain KSPGs endowed with cell binding properties [8]. Aside from KS principal function in cornea, this GAG type also participates in developmental biology, cellular signaling and migration, like the other GAGs chondroitin, dermatan and heparan sulfates [1]. For example, as opposed to chondroitin sulfates that are known to induced neurite growth and guide neurite migration during neural development, KS-containing molecules comprise a barrier to neurite growth in vitro [1,9,10]. KS and chondroitin sulfate seem to be working altogether, with opposite functions, to keep balanced the mechanisms involved in neural development. Nonetheless, in certain occasions, KS can also help direction of axons in neural development and regeneration in vivo [1,9,10]. 1.3. Scope of the review Due to the biological and medical roles of KS, as discussed above, as either naturally occurring functional molecules of the body, or as active ingredients in pharmaceutical formulations, a review paper outlining the major recent findings about this biological macromolecule seems to be relevant in the literature nowadays, especially, considering the rapid growing success of glycomics, and the importance of GAGs to this project. The inexistence of any review paper concerning KS in the literature of the last five years is an additional contributing factor to this publication. In this study, I discuss the major achievements made in the science of KS that have been deposited in the literature database within the 2010–2014 timeframe. This review is systematically divided into the following topics about KS: (i) its role in physiopathological conditions; (ii) as therapeutic or target molecule in diseases; (iii) methods of analysis and detection; and (iv) related enzymes, metabolism, and developmental biology. 2. Role in physiopathological conditions 2.1. Inflammation It is well-known that pro-inflammatory chemikones, and their consequential effects in leukocyte recruitment and activation, are at a first moment, regulated by GAGs found on cell surface PGs [11,12]. Based on this premise, Carlson and co-authors have investigated the role of KSPGs in the cornea as regulators of chemokine gradient and the neutrophil-dependent inflammatory process of this tissue [13]. The authors have postulated that KSPGs in cornea play a key role in controlling the chemokine gradient, its breakdown, and consequent resolution of the corneal inflammation. Experimentally, bacterial immunogenic lipopolisaccharide (LPS) was injected into the corneal stroma of mice. Extracts from this particular tissue were examined based on immunoblot methods. After 6 h of injection, while the amounts of 52 kDa protein core of keratocan were observed significantly reduced, the amounts of the 34/37 kDa products were proportionally increased. The appearance of these products of reduced molecular weights is coincident with the event of neutrophil infiltration in the corneal stroma.

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This appearance was not seen in explanted corneas or tissues from homozygote mutants for the CXCL1/KC (keratinocyte-derived chemokine) receptor (CXCR2−/− ). In these cases, the 34/37 kDa products and CXCL1/KC were fairly detected in the anterior chamber, into which the corneal stroma drains the products. Conversely, CXCL1/KC was seen highly elevated in homozygote mice mutants for keratocan and lumican. Based on these results, the authors have proposed a series of events to outline the KSPGs’ role in corneal inflammation. (i) Microbial infection of the corneal stroma (or exposure to bacterial products such as LPS) induces signaling in resident macrophage, and dendritic cells, leading to the production of CXC chemokines, including CXCL1/KC. (ii) GAG–chemokine interactions take place, and a chemokine gradient close to the inflamed tissue is formed for proper leukocyte attraction and activation. (iii) Neutrophils are recruited from peripheral vessels, activated and migrate along the chemokine gradient surface to the right site of infection for combating and defending the infected area. (iv) Activated neutrophils secrete metalloproteinases or stimulate the cornea for production of endogenous metalloproteinases. (v) Keratocan and lumican are cleaved, resulting thus KSPG-derived products of low molecular weights. (vi) These resultant fragments are drained by the adjacent corneal chambers and the inflammatory process is down-regulated. (vii) The normal condition of the tissue is restored. Ref. [13] has shown that KSPGs are not only exclusively involved in the hydration aspects of the corneal tissue, but also involved as major player in the inflammatory process of this tissue. In the work of Matsui and co-authors, the investigators have shown for the first time a negative correlation between the expression of KS by microglia and the progress of the experimental auto-immune neuritis (EAN) [14]. In this pathology, a demyelination process characterizes the auto-immunological response on cells of the peripheral nervous system. Matsui and coworkers had the goal of studying the role of KS in this immunological process [14]. They noticed that KS expression is fairly diminished in cases of EAN rat models. Microglia of the spine cord from normal rats are known to be the major cell type of the central nervous system involved with the expression of KS. In EAN models, however, microglia from the spine cord while increasing drastically the expression of pro-inflammatory cytokines, event of which is typical of the pathology, KS was observed considerably down-regulated. The reduced amounts of KS in this pathology make the condition of the patient harder to recover. By proving the negative association between EAN and the KS expression in microglia of EAN animal models, the authors suggested that low KS levels can be used as a marker for evaluate the status of the EAN disease. 2.2. Carcinoma of female genital tract Another work pointing the possible use of KS as a marker is Ref. [15]. In this reference, Miyamoto and coworkers have shown that KS can be immunohistochemically detected in carcinomas of the female genital tract (FGT). The authors have examined 102 samples of normal epithelia, and 110 samples of carcinomas from FGT (endometrium, cervix gland, cervix squamous, Fallopian tube, ovary follicle, ovary surface epithelium, and ectopic endometrium), from digestive organs (esophagus, stomach, small intestine, large intestine, liver, gallbladder, bile duct, and pancreas), from the urinary tract (kidney and urinary bladder) and from lung alveolus, lung bronchus, lung bronchial gland, mammary gland, thyroid, and mesothelium. In normal tissues, KS was consistently detected in the FGT and ectopic endometrium, but was not found in the digestive organs and urinary tract, and only partly detected in the lung, mammary gland and thyroid. In malignant tissues, while KS was consistently observed in carcinomas of the endometrium, ovary and fallopian tube, it was just partly detected in carcinomas of

the lung, mammary gland, thyroid, pancreas and mesothelium, and absent in carcinomas of the gastrointestinal tract, liver, and urinary tract. Conversely, KS was broadly found in carcinomas of the FGT, digestive organs and urinary tracts. Among these carcinomas, KS detection has suggested the possibility of diagnosing FGT carcinoma within 79.5% sensitivity, and 92.9% specificity. 2.3. Neural regeneration and plasticity Hilton and co-authors have made a recent study on the role of KS, and its digestive enzyme keratanase II (K-II), in the plasticity and recovery of the neural tissue after spinal cord injury (SCI) [16]. Supported by a previous work [17], the authors have concluded that KSPGs comprise an efficient inhibitor to the plasticity of the neural tissue after SCI [16]. Through the therapeutic point-of-view, the authors have questioned that if acute K-II treatments are able to enhance degradation of the KSPGs, these treatments could also enable consequential motor recovery by spinal regeneration. This question was investigated in the work [16], and the authors have thus suggested that the enzymatic therapy based on K-II should be implemented in future human trials of SCI patients. This is a key reference pointing out the relevance of KS-related enzymes in treatments of neural injury. This treatment option is highly desired since the regeneration of neural tissue is considerably limited if compared to the other tissues of the body. 2.4. Mucopolysaccharidosis Mucopolysaccharidosis is a class of diseases characterized by dysfunction in degrading and processing GAGs into their smallest structural components, the composing monosaccharides and free sulfate esters. This dysfunction results into inappropriate deposition of the unprocessed GAGs in cartilages. Mucopolysaccharidosis IVA (MPS IVA) also known as Morquio A syndrome, is particularly caused by the lack or deficiency in the galactose 6-sulfate sulfatase (GALNS). This enzyme is in charge of removing the 6-sulfation from the Gal units in KS chains during digestion. The simple abrogation of the 6-sulfation removal of Gal units impairs the further degrading steps of KS catabolism. This leads to a systemic skeletal dysplasia because of the excessive storage of KS in chondrocytes. In an effort better understand the clinical, biochemical and molecular mechanisms of MPS IVA, Dung and coworkers have examined numerous patients with MPS IVA [18]. This examination was carried out with the purpose of making a correlation between blood and urine KS levels with the phenotypes and genotypes of the patients as well as to attempt in determining a more precise prognostic and personalized treatment to these patients. Mutation screening of GALNS gene was undertaken by genomic PCR followed by direct sequential analysis. Blood and urine KS concentrations were noted to be age-dependent. Based on these biochemical parameters, MPS IVA patients considered with severe disease, showed higher concentrations of KS than those of attenuated conditions. This study has proven extensive allelic heterogeneity for the MPS IVA disease. Analyses based on accumulations of these mutations combined with measurements of plasma and urine KS concentration, permit accurate assessment of the severity and clinical stages of MPS IVA. These clinical analyses can be used to sort out the possible treatment options for MPS IVA [18]. 3. As a therapeutic or target molecule 3.1. As suppressor of cartilage damage The group of Dr. Ishiguro has published two papers in Biochemical and Biophysical Research Communications about the role of KS in suppressing the cartilage damage [19,20]. In the first paper of 2010,

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using a murine model of cartilage explants exposed to interleukin1␣ (IL-1␣), the authors were able to examine the potentiality of KS via intraperitoneal administration on the course of cartilage degradation [19]. Cartilage fragility was examined by measuring IL-1␣-induced aggrecan release from the cartilage explants treated or not with KS. The investigators have noticed that the aggrecan release was less in cartilage explants when KS is administered than in the cases where KS was not used. In the following paper of 2011, the authors have extended the knowledge about this potential KS medical role in cartilage damage using mice homozygote mutants for the enzyme N-acetylglucosamine 6-O-sulfotransferase (GlcNAc6ST) isoform 1 (GlcNAc6ST-1−/− ) compared to wild-type mice [20]. The authors used equivalent protocol of IL-1␣-induced KS release monitoring of explants as described in the previous paper [19]. In the later reference, they noticed as expected, that cartilage damage was higher in the doubly allelic mutant. This corroborates with the fact that naturally occurring KSPGs with the normal 6-sulfation content plays a crucial role in retarding the cartilage damage progression. The damage can be suppressed with intraperitonial injection of KS formulations, even at the mutant model, showing thus that exogenous KS can be used as a therapeutic molecule for combating cartilage degradation [20]. 3.2. As regulators of inflammation The situation described above about cartilage damage can be commonly seen in inflammatory processes like rheumatoid arthritis or osteoarthritis. Due to this reason, besides the protecting role of KS in models of cartilage damage, the authors of the two previous references have also investigated the role of this GAG in ameliorating inflammation process, particularly the IL-1␣-induced inflammation seen in rheumatoid arthritis [19,20]. In the first reference, by measuring arthritis score, body weight loss, and the histological conditions of joint cartilages of mice submitted or not to inflammatory conditions, the authors have seen that exogenous KS can ameliorate significantly the levels of the inflamed animals, in a direct correlation with the decreased release levels of aggrecan to the media. This last result indicates that KS treatment inhibits aggrecan release from cartilage in vivo. The authors speculate that KS ameliorates arthritis as a result of chondroprotection mechanism [19]. This same set of results was seen in in vivo mice models of GlcNAc6ST-1−/− . Although this mutation aggravated the IL-1␣induced inflammation in mice, the administration of exogenous KS can still reduce the pathology. Besides the beneficial effect in cartilage damage, the authors also indicate that KS must be treated as novel therapeutic compound for inflammation [20]. Shirato and coworkers have also demonstrated, publishing also at Biochemical and Biophysical Research Communications, the potential effect of KS in attenuating inflammatory events [21]. Rather than models of rheumatoid arthritis or osteoarthritis, this reference has used models of chronic obstructive pulmonary disease (COPD) [21]. The COPD is commonly characterized by inflammatory conditions driven by both immune cells and airway lung epithelial cells. This is consequence of bacterial and viral infections on the airways. KS has been postulated to be the major GAG type in airway secretions of the COPD patients. KS is known to be synthesized by the epithelial cells of airway surfaces in COPD conditions. COPD is expected to be the third leading cause of death in the World by 2020. Therefore, there is a pressing need for the development of new or additional treatments to this disease. The authors of Ref. [21] have shown that the KS disaccharide [Gal6S-(␤1–4)-GlcNAc6S], but not the N-acetyl lactosamine nor the chondroitin 6-sulfate disaccharides, is able to suppress the production of interleukin-8 stimulated by flagellin, a Toll-like receptor (TLR) 5 agonist commonly found in normal human bronchial epithelial (NHBE) cells. This flagellin inhibition leads to the impairment of phosphorylation

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of the epidermal growth factor receptor, and thus a downstream of the signaling pathway via TLR 5 in NHBE cells. These results indicate that fully sulfated KS disaccharide can be used, in a very near future, as a potent drug for prevention and treatments of the airway inflammatory conditions in COPD patients [21], especially those where bacterial infections are driving or aggravating the process. 3.3. In malignant cellular apoptotic process Burkitt’s lymphoma (also known as Burkitt’s tumor, Burkitt lymphoma, malignant lymphoma, or Burkitt’s type) is a cancer of the lymphatic system (in particular, B lymphocytes). The study of Nakayama and coworkers has shown the contribution of KSPGs, and its sulfation content to radio-resistance in human Burkitt’s lymphoma cellular apoptotic process [22]. The authors have induced apoptosis in Burkitt’s lymphoma cell lines by X-ray radiation at a dose rate of approximately 2.4 Gy/min. In order to understand the contribution of sulfation of KSPGs to this process, the authors studied the role of the sulfate donor transporters used in KS biosynthesis, the 3 -phosphoadenosine 5 -phosphosulfate transporter (PAPST), isoforms 1 (PAPST1) and 2 (PAPST2), and the GlcNAc6ST enzyme, isoforms CHST2, CHST6 and CHST7. The experiments were carried out in cell cultures submitted to different levels of radiation. The results have indicated the contribution of KSPGs, and the levels of GlcNAc 6-O-sulfation to reduce the radiation-induced apoptotic process in human Burkitt’s lymphoma cells. These findings collaborate to the future of radiation-based anticancer therapies of Burkitt lymphoma. In these cases, KSPGs could be taken as supplementary target molecules to improve the benefits of the radiation treatment. The combination of radiation with enzymes involved in degrading KS chains of the KSPGs (keratanases) is likely to enhance the quality, therapeutic levels, and outcomes expected during the therapy of this cancer. 3.4. As suppressors of amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is a motor neurondegenerative disease with various causes. It is characterized by muscle spasticity, rapidly progressive weakness due to muscle atrophy, difficulty in speaking (dysarthria), swallowing (dysphagia), and breathing (dyspnea). Hirano and coworkers have recently made the connection between the lack of KS and the increased development of ALS pathogenesis at its early phase [23]. The investigators have used comparatively both wild-type ALS-induced and GlcNAc6S−/− ALS-induced mice models, knocked-out just at their tissues of the central nervous system. Curiously, the doubly allelic mutant mice showed enhanced symptomatology of ALS, meaning a straight correlation of KS sulfation content with the progression of the disease. In ALS, KS expression is seen exclusively at a subpopulation of microglia of the wild-type mice. Its detection is reported to be only at early stages of the disease [23]. As opposed to the normal mice which showed a transient enhancement of microglia markers, this enhancement was considerably attenuated in GlcNAc6S−/− mutant mice. The KS expression by microglia was also observed in some human ALS cases. In all, Ref. [23] has indicated that KS plays an indispensable suppressive role in the early stages of development of the ALS pathogenesis. Although no tests of KS administration have been done, the authors still suggest the possibility of taking KS as a future drug candidate in therapeutic interventions against ALS disease. 4. Methods of detection and analysis Besides the extensive analytical work of Dr. Orii and co-authors in Ref. [18] from which genotype–phenotype correlations, biochemical and clinical parameters for MPS IVA diagnosis were

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established, especially on terms of the GALNS gene, the authors have also sophisticated the methods of liquid chromatography tandem–mass spectroscopy (LC/MS/MS), and sandwich ELISA to assess the KS levels in blood and urine of MPS IVA patients [24,25]. These works were carried out with the primary goal of establishing a secure and more precise methodology for the diagnosis of the disease. Through the analyses of numerous MPS IVA patients, classified into the categories as mentioned in the section 2.4, both methods intended to measure blood and urine KS levels were suggested to be fairly suitable for diagnosis, therapeutic monitoring, and longitudinal assessment of the disease progression. The LC/MS/MS method was seen to be able to measure over 10 times more KS present in body fluids than the ELISA method. Lettry and coworkers have evaluated a new system to identify cartilage turnover and/or degradation via detection and measurement of a KS in equine sera [26]. Serum samples of fifteen 1-year-old and fifteen 2-year-old horses were analyzed in terms of the usual epitope of KS detected by the monoclonal antibody 5D4, and by the new high-sensitive keratan sulfate (HSKS) antibody. The reaction of detectability using both antibodies (5D4 and HSKS) was performed using ELISA. The results have indicated that age has no effects on the measurements with 5D4, as opposed to HSKS which was able to detect higher amounts of KS in the 2-year-old horses. This indicates that HSKS, but not 5D4, is able to detect early signs of cartilage metabolic changes in horses [26]. The HSKS specificity is likely to be useful for human sera too. Mapping the binding protein partners of KS is a secure way to understand the possible biological roles of KS. For example, embryonic corneal keratocytes, and sensory nerve fibers grow and differentiate according to chemical cues they interact with in the corneal stroma ECM. As already discussed, KS is an abundant GAG type of the corneal ECM. Conrad and coworkers have thus investigated which proteins are able to interact with KS in this tissue, and then regulate the migration of keratocytes and corneal nerve growth [27]. To achieve their objective, biotinylated KS was prepared and used in a microarray approach with a multiple protein. This procedure helps the assessment of possible GAG–protein interactions involved in nerve growth or keratocyte migration. Besides binding to certain kinases, cytoskeletal, membrane and secreted proteins as well as many proteins involved with nerve function, highly sulfated KS was also able to interact with ECM proteins such as SLIT 2 ROBOs, ephrins, semaphorins, and two nerve growth receptor types [27]. This reference has successfully shown that microarray applied to a vast and diverse set of proteins is efficiently able to map the possible binding protein partners of KS. The list of interacting partners from this study is also helpful to understand their biological roles. Recently, we have shown that KS could be detected in pharmaceutical chondroitin sulfate formulations destined to oral administration, in a percentage up to 16% of the total active ingredient [28]. The KS detection was not possible solely on the basis of horizontal electrophoretic analysis as predicted and recommended by the international pharmacopeias. But, primarily by liquid-state nuclear magnetic resonance (NMR) spectroscopy combined with high performance liquid chromatography (HPLC) techniques, the detection turned to be feasible. Besides showing the proper analytical methods to be used to detect KS in chondroitin sulfate formulations, this reference has also forewarned the standard pharmacopeias to update their monographs and alerted the manufacturers and supervisory agencies regarding this contamination. Even though the contaminant was KS, an inert and harmless compound, the amounts of the active ingredient chondroitin sulfate specified in the label claims of the analyzed products were completely far off the expected [28]. This is a reference showing the potentiality of the proper method chosen for analysis of a GAGbased material, particularly KS and chondroitin sulfate.

The study of Nakano and coworkers aimed at detecting KS products containing mechanically separated chicken meat (MSCM) with cartilage particles [29]. To accomplish the analytical work, drydefatted samples of MSCM and meat products, containing or not MSCM, were digested with papain and non-dialyzable fractions of each digestion were examined by immunodiffusion analysis using an anti-KS monoclonal antibody belonging to the IgM family. Antibody-based detection was negative for all samples of meat products without MSCM, while the samples of MSCM and all samples of meat products containing MSCM gave clear reaction with the antibody. The immunodiffusion test described at this reference seems to be a very simple and sensitive method for qualitative analysis of KS, especially in case of detection of KS in MSCM-included meat products [29]. Weyers and co-authors have investigated commercially available and isolated KS samples, both of bovine cornea origins, in order to prove equality of the samples [30]. While the structural analyses were based on size-exclusion chromatography, NMR spectroscopy, HPLC–MS, polyacrylamide gel electrophoresis (PAGE), the functional analyses were based on surface plasmon resonance (SPR) to assess the capabilities of these two KS materials in interactions with sonic hedgehog, fibroblast growth factor-1, and fibroblast growth factor-2. The data have pointed toward that both natural and commercial sources give rise to KS materials of equivalent structural and functional properties. This conclusion together with the description of a purification method capable of extracting multi-milligram quantities of bovine corneal KS have indicated that isolation and purification of KS material is a secure way to generate KS amounts when the shipment or commercial availability of this product is restricted [30]. After our publication reporting the KS amounts in pharmaceutical chondroitin sulfate formulations [28], other publications describing methods to either detect KS or to selectively remove it from the chondroitin sulfate preparations have been appeared in the literature [31,32]. The method for specific and sensitive detection of KS in chondroitin sulfate formulation is based on a combination of immunodiffusion analysis and ELISA, both using anti-KS monoclonal antibody of the family IgM [31]. The immunodiffusion method and the anti-KS antibody are the same of those reported in Ref. [29]. In Ref. [31], the authors discussed that the immunodiffusion method is a quick one-step procedure for accurate detection of KS. And ELISA is a very reliable method to quantify the amounts of the unexpected KS material in chondroitin sulfate formulations. The group of Prof. Volpi has shown that KS can be selectively removed from pharmaceutical chondroitin sulfate formulations by means of sequential precipitation with ethanol [32]. The authors have reported that KS in 10–15% of KS-contained chondroitin sulfate preparations can be selectively precipitated by the presence of increasing percentages of saturated ethanol (reagentgrade). Chondroitin sulfate formulations can be reached in almost 100% pureness if 1.0 volume of ethanol is added to the samples containing KS. The authors stated that precipitation of KS based on increasing percentages of ethanol can be considered a reliable and simple industrial preparative method to produce chondroitin sulfate devoid of residual KS amounts originated from imperfect steps of large-scale isolation and purification of chondroitin sulfate from shark cartilage tissues [32]. Antibodies can recognize non-, mono-, and di-sulfated epitopes with different levels of interaction. Despite the wealth of information of NMR and sensitive of MS, the exploration of specific antibodies that interact with KS, as well as with differentially sulfated regions of KS backbone, seems to be the manner mostly used for detection of KS and its structural motifs. Among many references studied in this review reporting the use of anti-KS antibodies, the work of Kawabe and coworkers has shown the most specific antibody to be used against KS [33]. The anti-KS antibody reported

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by them recognizes KS chains lacking oversulfated structures. This antibody can be used in human induced pluripotent stem cells as well as embryonic stems cells. This antibody can be used as a new molecular probe for disclosing the roles of KS in these cell types.

5. KS-related enzymes, synthesis and developmental biology It is well-known that ECM molecules, in particular GAGs such as KS, are involved in remodeling tissues and morphogenesis during the embryonic development and stages of growth. In order to evaluate the structural changes of KS and its correlation to the changes observed during the development of corneal stroma (it is worth mentioning again that KS is very relevant to the functionality and development of corneal stroma), Liles and coworkers have used anti-KS antibodies of differential specificities for the KS sulfation contents in ELISA tests. These tests were combined with dosages of ECM collagen fibrils by synchrotron X-ray diffraction analyses and hydroxyproline measurements to assess respectively the variations of sulfation in KS, and the deposition of collagen in to chick cornea tissues. The chick models used were embryonic specimens with 12–18 days of incubation [34]. The investigators have observed a significant increase on the sulfation content of KS in the models from the 12th and 18th days. A directly increase on the hydroxyproline content, and a decrease of the collagen interfibrillar distance were also noted. The authors have speculated that these structural changes on the ECM compounds of the chick embryos over the time are crucial to regulate the levels of hydration and the molecular architecture of the studied tissue during the development of the animal [33]. These changes are likely to be expected in human embryonic development. As mentioned in Section 3.3, the carbohydrate sulfotransferase 6 (CHST6), a functional enzyme involved in the biosynthesis of KS, is responsible for proper sulfation of this GAG. Macular corneal dystrophy is a disorder characterized by a rare autosomal recessive mutation in the gene of this enzyme. The synthesis of unsulfated or less sulfated KSPGs in the corneal cells leads to visual impairment, the main characteristic of macular corneal dystrophy as mentioned in Section 1.2. The work of Di Iorio and coworkers had the primary goal identifying which corneal cells are able to express CHST6 and KSPGs in order to exert the activities in cornea [35]. This understanding could help the cases of therapeutic interventions for macular corneal dystrophy. The authors have used immunohistochemistry, semiquantitative real time-PCR, in situ RNA hybridization, and HPLC analyses. Both the expression and localization of CHST6, KS and protein cores of KSPGs (particularly mimecan and lumican) were analyzed in human cornea sections, and in cultured primary keratinocytes and keratocytes. When analyzing the human cornea sections, KS was predominantly found at the stroma, and barely detectable at the epithelial region. This similar pattern was observed for the epidermis, in which KS is mainly synthesized by derma layers. Expectedly, the CHST6 expression was noted to be higher at the suprabasal, and not at the basal epithelial layers. The expression was also significant in the stroma, and in the endothelial portions of the cornea. In agreement with this set of in vivo results, cultured stromal keratocytes were noted able to express and secrete more KS amounts than keratinocytes. The authors have inclusively pointed out that stromal keratocytes were the primary cells involved in the CHST6 expression in the corneal stroma, as well as in the syntheses of KSPGs with normal levels of sulfation like lumican and mimecan. As opposed to keratocytes, corneal keratinocytes do not express lumican and mimecan, and express little CHST6. The contribution of this reference on mapping the locations and cell types involved in the expression of CHST6 as well as in the synthesis of properly sulfated KSPGs is big in terms of

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medical intervention. From this work, the authors have suggested that any curing or therapeutic treatments to fight against the visual disorder macular corneal dystrophy must be aimed at the stromal keratocytes rather than inefficiently aimed at the other tissues or corneal cells [35]. In the publication of Maszczak-Seneczko and co-authors at the Journal of Biological Chemistry of 2013, the investigators have clearly showed, as expected, that not only the enzymes involved in the biosynthesis of KS are relevant to the structural integrity and expression of this GAG type, but also those involved in sugar transport such as the UDP-N-acetylglucosamine transporter, SLC35A3 [36]. The investigators of this work have silenced SLC35A3 in cell lines of Mardin–Darby canine kidney, and they noted a drastic decrease in the content of KS while no changes in the biosynthesis of heparan sulfate have occurred. Although heparan sulfate is also made up of N-acetyl glucosamine units in its disaccharide repeating building blocks, the SLC35A3 transporter seems to be primarily used to KS biosynthesis. This could be true if we consider the different biosynthetic routes of KS and heparan sulfate. While the latter is synthesized as a regular PG, KS is commonly synthesized as N- or O-glycans. Indeed, the authors of the work [36] have also noted that, besides KS, the silence of SLC35A3 additionally leads to depletion of highly branched multi-antennary N-glycans. As discussed in Section 1.1, chain modification of KS relies on sulfation at C6-positions of any of its composing units. In Section 3.1, GlcNAc6ST, in particular isoform 1 (GlcNAc6ST1), was reported to be the main sulfotransferase involved in 6-sulfation of the GlcNAc units of KS [20]. In Ref. [26], the authors have reported the use of an anti-KS 5D4 antibody against the Gal6S-GlcNAc6S epitope of KS. In a recent work of Hoshino and co-authors, the other sulfotransferase KSGal6ST, responsible to place sulfation at the 6C-position of Gal units in KS, was also examined in terms to its specificity with 5D4 reactivity in developing brains [37]. By examining brains of GlcNAc6ST1- or KSGal6ST-deficient mice at postnatal periods, the authors were able to observe that the 5D4 epitope, formed primarily in the cortical marginal zone, subplate and dorsal thalamus, was completely eliminated in any cases. Hence, a cooperative expression of both sulfotransferases is required for the proper neural development in mice, as seen by the immunoreactivity of the 5D4 KS epitope [37].

6. Major conclusions and future prospects In this review I have compiled most of the information about KS available from the publications deposited in the scientific literature database in the time window of 2010–2014. The latest review article concerning KS dates from 2006 [38], and its discussion is primarily focused on function of KS at the neural system. The latest general review discussing the major information about structure, function and biosynthesis of KS dates from 2000 [1]. Inasmuch, this current review seems to be timely, especially considering the growing relevance of the currently ongoing glycomic era [39] and the significant contribution of GAGs to glycomics. Here, I decided to not overlap discussion with the previous reviews. Thus, I have focused the discussion of this publication on the most achievements made in the science of KS during the last five years. This review analyzed the references systematically divided into four major topics. In the section discussing the first topic, the roles and effects of KS, and related enzymes, have been examined in perspective of their physiologic and medical functions, especially in inflammation, in carcinoma of the female tract, in neural regeneration and plasticity, and in mucopolysaccharidosis. In inflammation, KSPGs seem to regulate the gradient of chemokines responsible to attract, guide, and activate the leukocytes. In auto-immune neuritis, KS is curiously down-regulated, even though pro-inflammatory chemokines

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Table 1 Physiopathological roles and medical functions of KS. System

Function

Reference

Cornea physiopathology

KS, as constitutive component of cornea, when administrated as functional ingredient in eye drops, allows health improvement in macular corneal dystrophy and keratoconus.

[1,6,9]

Neural development and pathology

While in certain conditions, KS impairs neurite growth in vitro, in other conditions directs axons in neural development and regeneration in vivo. KS impairs plasticity and recovery of spinal cord injury. Its degradation using keratanase suggests a therapeutic avenue.

[1,9,10]

KSPGs control chemokine gradient formation and regulate neutrophil migration. Administration of KS leads to amelioration in inflammatory processes, especially in rheumatoid arthritis and chronic obstructive pulmonary disease. KS is seen fairly diminished in microglia of spine cord (main tissue of the central nervous system responsible for KS synthesis) during auto-immune neuritis.

[13] [19–21]

Cancer biology

KS can be used as a biomarker in carcinomas of female genital tract. KS and its sulfation content reduce radio-induced apoptotic process in human Burkitt’s lymphoma cells. Enzymatic therapy using KS-related degrading enzymes is a route to fight this pathology.

[15] [22]

Mucopolysaccharidosis

In MPS IVA, the enzyme galactose 6-sulfate sulfatase is somewhat compromised. This leads to deposition of KS in chondocytes. Enzymatic therapy using KS-related degrading enzymes is a route to fight this pathology.

[18]

Cartilage physiopathology

KS, as constitutive component of cartilage, when intraperitonially administered in rat models can impair or delay cartilage damage.

[19,20]

Amyotrophic lateral sclerosis

The clinical use of KS leads to suppression of the early developing stages of this disease.

[23]

Inflammation

are expectedly found at increased levels. The low amounts of KS in this pathology can make the ill condition even worst. In carcinoma of the female tract, KS, which is broadly found, can be used as potential biomarker to this type of cancer. In neural regeneration and plasticity, although KS seems to play a negative role in inhibiting the recovery process after spinal cord injury, the use of the digestive enzyme, keratanase II seems to be of therapeutic usefulness. Since KS is inadequately deposited in patients of MPS IVA due to dysfunction of the galactose 6-sulfate sulfatase, it has been reported that measurements of the accumulated mutation on the gene GALNS of this enzyme, as well as the plasma and blood KS concentrations, are quite useful to the diagnostic of the severity levels of this disease. Curiously, all works available in the literature between 2010 and 2014 concerning the beneficial roles of KS to health or as target molecule for therapeutic purpose come from Japan [19–23]. These works have been revisited in the second topic (Section 3) of this review, and are describing the potential roles of KS in suppressing the cartilage damage, in combating inflammation process, in reducing radiation-induced apoptosis of lymphoma cells, and in suppressing early phase pathogenesis of ALS. The roles of KS studied in Sections 2 and 3 are summarized in Table 1 for simplification and straightforward notation. Section 4, discussing the third topic, discoursed about main methods used for analysis and detection of KS. They include liquid chromatography, especially those based on high-performance automated system; analyses of genes related with biosynthetic and metabolic enzymes of KS; mass spectroscopy; immune-based methods like ELISA, immunodiffusion analysis and interactions with specific antibodies against KS or with differential structural epitopes of this GAG type; liquid-state NMR spectroscopy; surface plasmon resonance; both horizontal and vertical electrophoretic methods; and microarray using multiple possible ligands of KS. It is worth mentioning that the immune-based analytical methods comprise the mostly used methods, perhaps, due to commercial availability of the anti-KS antibodies. The last section of the review (Section 5) has analyzed the contribution of references concerning KS in terms of its related enzymes, metabolism and roles in developmental biology. From these references, it has been reported that the structural integrity of KS and consequently its biological roles depends primarily on the proper functions of the enzymes KSGalST, SLC35A3, and GlcNAc6ST

[16]

[14]

(particularly the CHST6 isoform). The natural changes on the KS structure seen during the embryogenesis, especially at the early days of development, were reported to be a crucial onset to the adequate corneal growth and functionality. The main structural change observed in this early period of embryonic development was the increase of sulfation content in KS. This increase is directly related with the expression and function of biosynthetic enzymes related with sulfation of KS, in particular CHST6. Although research about the medical and physiopathological roles of KS is vast as documented here, very little is investigated in the field of structural glycobiology of this GAG, especially by the most powerful analytical technique available in the field, solution NMR spectroscopy. For instance all GAGs, except KS, have been studied in what concerns their conformational [38] and dynamical [39] behaviors free in solution, or at their bound-states in intermolecular complexes with proteins [40]. The lower dedication to these fields of structural glycobiology of KS might be associated to the lower abundance of this GAG type as compared to the availability of the other ones. I believe that future investigations of KS by solution NMR experiments such as residual dipolar coupling and NOE measurements for conformational studies; spin-relaxation measurements and assignments of changes in scalar coupling constants and chemical shifts for dynamical studies; and finally, saturation transfer difference and transferred NOE for understanding the properties of KS in complexes with the protein partners in solution will definitely collaborate in favor to the growth of the science of KS. These NMR-based studies have been largely and successfully used for the other GAG types [40–42]. This same success must now be turned to KS. Conflict of interest The author states that he is not aware of any authorship, affiliations, memberships, funding, or financial holdings that might be perceived as damaged or as affecting the objectivity of the content of this material. The author declares no conflict of interest by any part. Funding This study was supported by grants Universal-14/2013[470330/2013-9] from Conselho Nacional de Desenvolvimento

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Keratan sulfate: an up-to-date review.

Keratan sulfate (KS) is a glycosaminoglycan (GAG) type consisted of a sulfated poly-N-acetyl lactosamine chain. Besides acting as a constitutive molec...
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