cells Article Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using the Keratanase Generated BKS-1(+) Neoepitope: Correlation of Expression Patterns with the Class II SLRPs, Lumican and Keratocan Anthony J. Hayes 1 and James Melrose 2,3,4,* 1 Bioimaging Research Hub, Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3AX, Wales, UK; HayesAJ@cardiff.ac.uk 2 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia 3 Raymond Purves Laboratory, Institute of Bone and Joint Research, Kolling Institute of Medical Research, Northern Sydney Local Health District, Faculty of Medicine and Health, University of Sydney Royal North Shore Hospital, St. Leonards, NSW 2065, Australia 4 Sydney Medical School, Northern, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW 2065, Australia * Correspondence: [email protected] Received: 8 February 2020; Accepted: 28 March 2020; Published: 30 March 2020 Abstract: This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development. Keywords: keratan sulfate; keratocan; lumican; intervertebral disc; spinal cord; endochondral ossification; neural development 1. Introduction Keratan sulfate (KS) is a widely distributed glycosaminoglycan (GAG) component of proteoglycans (PGs) in the extracellular matrix (ECM) and on cell surfaces of tensional and weight-bearing connective tissues such as ligaments, tendons, articular cartilage, and intervertebral discs (IVD), in addition to corneas and the central and peripheral nervous systems (CNS/PNS) [1–3]. Historically, most of the Cells 2020, 9, 826; doi:10.3390/cells9040826 www.mdpi.com/journal/cells Cells 2020, 8, x FOR PEER REVIEW 2 of 18 bearing connective tissues such as ligaments, tendons, articular cartilage, and intervertebral discs Cells 2020, 9, 826 2 of 17 (IVD), in addition to corneas and the central and peripheral nervous systems (CNS/PNS) [1–3]. Historically, most of the information on the spatio-temporal distribution and function of KS-PGs has informationbeen gleaned on through the spatio-temporal the use of the distribution monoclonal and antibody function of(MAb) KS-PGs 5-D has-4 beenwhich gleaned detects through disulfate thed useheptasaccharide of the monoclonal regions antibody of high (MAb) charge 5-D-4 density which in detects KS chains disulfated [4]. KS heptasaccharide is initially synthesized regions of highas a chargepolylactosamine density in chain KS chains sulfate [4d]. on KS its is GlcNAc initially synthesizedresidues in a asmono a polylactosaminesulfated KS glycoform chain sulfated [5], and on with its GlcNActissue maturation residuesin galactose a monosulfated residues KSof the glycoform GlcNAc [-5Gal], and KS withrepeat tissue disaccharide maturation become galactose progressively residues ofsulfate the GlcNAc-Gald at C6 leading KS repeatto regions disaccharide of disulfation become on progressivelythe KS chains. sulfated KS is heterogeneous at C6 leading and to regions contains of disulfationvariably distributed on the KS chains.regions KS which is heterogeneous are non-sulfate and containsd, and variably mono- distributedand disulfate regionsd [1 which]. While are non-sulfated,heptasaccharide and groupings mono- and of disulfated disulfated [ 1regions]. While on heptasaccharide KS are detected groupings by MAb of5- disulfatedD-4, this antibody regions onprovides KS are no detected information by MAb on 5-D-4,mono- thisor non antibody-sulfate providesd regions no of informationKS [6]. As MAb on mono-or 5-D-4 is non-sulfatedincapable of regionsdetecting of KSlow [ 6charge]. As MAb density 5-D-4 KS is incapablespecies, its of application detecting low to chargeembryonic density and KS fetal species, connective its application tissues tomay embryonic under represent and fetal connectivethe quantity tissues and maydistribution under represent of KS GAGs the quantity which andhave distribution yet to develop of KS GAGsappreciable which levels have of yet highly to develop sulfate appreciabled KS epitopes. levels Antibodies of highly to sulfated the mono KS- epitopes.sulfated (MAb Antibodies R10-G, to 1B4) the mono-sulfated[7] and non-sulfate (MAbd R10-G, polylactosamine 1B4) [7] and non-sulfatedregions of KS polylactosamine (MAb ‘i’) [8] regions are now of KSavailable (MAb ‘i’) and, [8] arein nowcombination available with and, specific in combination KS depolymerizing with specific KSenzymes, depolymerizing functional enzymes, roles for functional these regions roles forare thesenow regionsemerging are [9 now]. BKS emerging-1 immunolocalization [9]. BKS-1 immunolocalization is therefore a more is therefore accurate a quan moreti accuratetative measure quantitative of KS measuresince one ofBKS KS-1 since epitope one is BKS-1 labeled epitope per KS is chain labeled in all per KS KS glycoforms chain in allirrespective KS glycoforms of their irrespective charge status of their(Figure charge 1). status (Figure1). FigureFigure 1.1. Structure of a typical KS-IKS-I chain and identificationidentification of high charge density 5-D-45-D-4 andand lowlow chargecharge densitydensity R10GR10G andand 1-B-41-B-4 antibodyantibody bindingbinding sitessites ((aa)) inin N-N- ((bb)) andand O-linkedO-linked KSKS chainschains ((cc).). MAbMAb BKS-1BKS-1 identifiesidentifies KSKS inin earlyearly tissuetissue developmentdevelopment inin KS-PGsKS-PGs containingcontaining lowlow sulfationsulfation KSKS glycoformsglycoforms asas wellwell asas highlyhighly sulfatedsulfated KSKS inin maturemature tissues.tissues. In corneal tissues, MAb 5-D-45-D-4 and BKS-1BKS-1 detectdetect similarsimilar cornealcorneal KS-PGKS-PG populationspopulations byby WesternWestern blotting blotting [ 10[10]] and, and, in in immunolocalizations, immunolocalizations, BKS-1 BKS- displays1 displays a morea more subtle subtle localization localization of of KS KS than than 5-D-4 5-D-4 [11 [11],] possibly, possibly due due to to the the stoichiometry stoichiometry of of 5-D-4 5-D- and4 and BKS-1 BKS-1 antibody antibody binding binding to to KSKS chains.chains. BKS BKS-1-1 staining staining is is quantitative, quantitative, thus thus the the relative relative levels levels of ofKS KS in indifferent different regions regions of a of given a given tissue tissue can canbe precisely be precisely compared. compared. Furthermore, Furthermore, whereas whereas MAb MAb 5-D- 5-D-44 detects detects longer, longer, more more highly highly sulfate sulfatedd KS chains, KS chains, BKS-1 BKS-1 detects detects shorter, shorter, less highly less highlysulfated sulfated KS chains, KS chains,typical typicalof those of thosefound found in lumican, in lumican, keratocan keratocan,, and and proline/arginine proline/arginine-rich-rich end end leucine leucine-rich-rich repeat proteinprotein (PRELP)(PRELP) inin fetalfetal oror embryonicembryonic tissues.tissues. The presence ofof these low sulfationsulfation KSKS glycoforms hashas previouslypreviously beenbeen under-appreciated under-appreciated [1 ].[1 For]. For example, example, a mono-sulfated a mono-sulfate KSd glycoform KS glycoform in electro-sensory in electro- tissuessensory of tissues the Ampullae of the Ampullae of Lorenzini of Lorenzini in elasmobranch in elasmobranch sharks and sharks rays and is the rays sole is GAGthe sole found GAG in found these tissuesin these [12 tissues] and is[12 the] and most is sensitivethe most protonsensitive detection proton agentdetection known agent in natureknown [ 13in]. nature A natural [13]. killer A natural (NK) cell-restrictedkiller (NK) cell KS-glycoprotein-restricted KS- (PEN5),glycoprotein a developmentally-regulated (PEN5), a developmentally post-translational-regulated post modification-translational of the platelet selectin glycoprotein ligand-1 (PSGL-1), is a unique binding site for L-selectin expressed Cells 2020, 9, 826 3 of 17 by platelets, endothelial cells, and leukocytes serving as a