Keratan Sulfate Proteoglycan Research Articles

Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea’s collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

Transcriptome and proteome revealed the differences in 3 colors of earlobe in Jiangshan Black-bone chicken

The earlobe is a featherless, exposed thickening located beneath the ear canal of chickens, which plays a visual signaling role in age, performance, mental vitality, reproduction, and other aspects. However, despite its importance, there have been few studies on the color differences and formation mechanisms of chicken earlobes, particularly the structurally blue earlobes characteristic of the Jiangshan black-bone chicken. In this study, we explored the physiological mechanisms that may influence the formation of differently colored earlobes using 3 types of earlobes from Jiangshan black-bone chickens: light peacock green (Green group), dark peacock green (Blue group), and dark reddish purple (Black group). All 3 earlobe colors exhibited positive melanin Masson-Fontana staining, and the thickness of collagen fibers in the dermis decreased in the order of Green, Blue, and Black groups. A total of 1,953 differentially expressed genes (DEGs) were detected in the 3 earlobes through mRNA sequencing, among which the GO term "collagen trimer" was significantly enriched in DEGs between groups. Additionally, 716 differentially expressed proteins (DEPs) were identified in the 3 earlobes using 4D-DIA proteomics, with the term "collagen fibril organization" being significantly enriched in DEPs between the Green and Black groups. Integrated analysis of transcriptome and proteome data revealed that 12 DEGs and DEPs were commonly differentially expressed between the Green and Black groups, including the gene LUM (corneal keratan sulfate proteoglycan), which was significantly enriched in the "collagen fibril organization" GO term. In conclusion, our study suggests that LUM plays a crucial role in the formation of peacock green earlobes in Jiangshan black-bone chickens. The high level of LUM in peacock green (Green and Blue groups) may affect collagen nanostructures, leading to a stronger effect of melanin-supported dermal collagen on the production of non-iridescent structural colors through coherent scattering, resulting in a bright structural blue color in Jiangshan black-bone chickens. In contrast, the low expression of LUM in dark reddish purple (Black group) reduces the reflection of non-iridescent structural colors, making the earlobe color appear almost black, similar to melanin.

The versatile roles of lumican in eye diseases: A review

Lumican is a keratan sulfate proteoglycan that belongs to the small leucine-rich proteoglycan family. Research has lifted the veil on the versatile roles of lumican in the pathogenesis of eye diseases. Lumican has pivotal roles in the maintenance of physiological tissue homogenesis and is often upregulated in pathological conditions, e.g., fibrosis, scar tissue formation in injured tissues, persistent inflammatory responses and immune anomaly, etc.Herein, we will review literature regarding the role of lumican in pathogenesis of inherited congenital and acquired eye diseases, e.g., cornea dystrophy, cataract, glaucoma and chorioretinal diseases, etc.

Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy.

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.

Keratan sulfate proteoglycan as an axonal insulating barrier in the forebrain of fetuses with alobar/semi-lobar holoprosencephaly.

Keratan sulfate (KS) is an abundant proteoglycan in the developing human CNS where it functions as an extracellular axonal guidance molecule, repelling glutamatergic while facilitating GABAergic axons. It ensheaths axonal fascicles. In fetal brain maturation, KS acts as a barrier to axonal penetration. Its possible role in the pathogenesis of fetal holoprosencephaly (HPE) was studied. Forebrains of 6 human fetuses with HPE identified by prenatal ultrasound were examined at autopsy with KS immunoreactivity and other markers of cellular maturation and synaptogenesis, with age-matched controls. KS was strongly expressed in astrocytes in the thalamus from 13 weeks gestational age (GA) and in globus pallidus but not corpus striatum. Cortical plate reactivity was limited to the molecular zone, where KS was excessive, ensheathing individual transverse molecular zone axons. Axonal envelopment preceding myelination also was seen in the internal capsule and thalamocortical projections, but perifascicular KS was diminished. KS was not expressed in hippocampus in either HPE or controls. Glutamate receptor-2 (GluR2) was evident in hippocampal granular and pyramidal neurons at mid-gestation. KS distribution did not, however, correlate with synaptophysin. Excessive ensheathment of axons by KS provides additional protection of GABAergic inhibitory axons and synapses that may help suppress epileptogenesis. Though involved in selection of excitatory and inhibitory synaptogenesis, KS does not follow a developmental sequence corresponding to synaptophysin or GluR2 reactivities in either HPE or in normal fetal brain.

Structural insight into the binding of human galectins to corneal keratan sulfate, its desulfated form and related saccharides

Glycosaminoglycan chains of keratan sulfate proteoglycans appear to be physiologically significant by pairing with tissue lectins. Here, we used NMR spectroscopy and molecular dynamics (MD) simulations to characterize interactions of corneal keratan sulfate (KS), its desulfated form, as well as di-, tetra- (N-acetyllactosamine and lacto-N-tetraose) and octasaccharides with adhesion/growth-regulatory galectins, in particular galectin-3 (Gal-3). The KS contact region involves the lectin canonical binding site, with estimated KD values in the low µM range and stoichiometry of ~ 8 to ~ 20 galectin molecules binding per polysaccharide chain. Compared to Gal-3, the affinity to Gal-7 is relatively low, signaling preferences among galectins. The importance of the sulfate groups was delineated by using desulfated analogs that exhibit relatively reduced affinity. Binding studies with two related di- and tetrasaccharides revealed a similar decrease that underscores affinity enhancement by repetitive arrangement of disaccharide units. MD-based binding energies of KS oligosaccharide-loaded galectins support experimental data on Gal-3 and -7, and extend the scope of KS binding to Gal-1 and -9N. Overall, our results provide strong incentive to further probe the relevance of molecular recognition of KS by galectins in terms of physiological processes in situ, e.g. maintaining integrity of mucosal barriers, intermolecular (lattice-like) gluing within the extracellular meshwork or synaptogenesis.

N-glycosylation of the human \u03b21,4-galactosyltransferase 4 is crucial for its activity and Golgi localization

β1,4-galactosyltransferase 4 (B4GalT4) is one of seven B4GalTs that belong to CAZy glycosyltransferase family 7 and transfer galactose to growing sugar moieties of proteins, glycolipids, glycosaminoglycans as well as single sugar for lactose synthesis. Herein, we identify two asparagine-linked glycosylation sites in B4GalT4. We found that mutation of one site (Asn220) had greater impact on enzymatic activity while another (Asn335) on Golgi localization and presence of N-glycans at both sites is required for production of stable and enzymatically active protein and its secretion. Additionally, we confirm B4GalT4 involvement in synthesis of keratan sulfate (KS) by generating A375 B4GalT4 knock-out cell lines that show drastic decrease in the amount of KS proteoglycans and no significant structural changes in N- and O-glycans. We show that KS decrease in A375 cells deficient in B4GalT4 activity can be rescued by overproduction of either partially or fully glycosylated B4GalT4 but not with N-glycan-depleted B4GalT4 version.

Proteoglycans in Obesity-Associated Metabolic Dysfunction and Meta-Inflammation.

Proteoglycans are a specific subset of glycoproteins found at the cell surface and in the extracellular matrix, where they interact with a plethora of proteins involved in metabolic homeostasis and meta-inflammation. Over the last decade, new insights have emerged on the mechanism and biological significance of these interactions in the context of diet-induced disorders such as obesity and type-2 diabetes. Complications of energy metabolism drive most diet-induced metabolic disorders, which results in low-grade chronic inflammation, thereby affecting proper function of many vital organs involved in energy homeostasis, such as the brain, liver, kidney, heart and adipose tissue. Here, we discuss how heparan, chondroitin and keratan sulfate proteoglycans modulate obesity-induced metabolic dysfunction and low-grade inflammation that impact the initiation and progression of obesity-associated morbidities.

Osteoadherin serves roles in the regulation of apoptosis and growth in MC3T3‑E1 osteoblast cells.

Small leucine‑rich proteoglycans (SLRPs) are a class of proteoglycans that are characterized by small protein cores and structures of leucine‑rich repeats. SLRPs are expressed in most extracellular matrices and share numerous biological functions that are associated with binding of collagens and cell surface receptors. Osteoadherin (also termed osteomodulin) is encoded by the Omd gene and is a keratan sulfate proteoglycan of the class II subfamily of SLRPs. Osteoadherin is highly expressed in mineralized tissues, including bone and dentin; however, it's precise roles remain unknown. The present study determined the Omd expression levels and investigated the effects of over‑ and under‑expression of osteoadherin in osteoblastic cells. Omd mRNA expression increased with osteoblast differentiation in MC3T3‑E1 cells. In C2C12 cells, Omd mRNA expression was induced by bone morphogenetic protein (BMP)2. Reporter assays similarly demonstrated activation of the Omd gene promoter following co‑transfection with Smad1 and Smad4, which are intracellular signaling molecules of the BMP2 signaling pathway. Overexpression of Omd increased the viability and decreased caspase 3/7 activity in MC3T3‑E1 cells. By contrast, following transfection with small interfering RNA for Omd, viable cell numbers were decreased and caspase 3/7 activity was increased. Furthermore, overexpression of Omd reduced the expression of CCN family 2 in these cells. These results demonstrate that Omd expression is regulated during osteoblast differentiation, and that the protein product osteoadherin serves roles in the apoptosis and growth of osteoblast cells.

Functional Consequences of Keratan Sulfate Sulfation in Electrosensory Tissues and in Neuronal Regulation.

Keratan sulfate (KS) is a functional electrosensory and neuro-instructive molecule. Recent studies have identified novel low sulfation KS in auditory and sensory tissues such as the tectorial membrane of the organ of Corti and the Ampullae of Lorenzini in elasmobranch fish. These are extremely sensitive proton gradient detection systems that send signals to neural interfaces to facilitate audition and electrolocation. High and low sulfation KS have differential functional roles in song learning in the immature male zebra song-finch with high charge density KS in song nuclei promoting brain development and cognitive learning. The conductive properties of KS are relevant to the excitable neural phenotype. High sulfation KS interacts with a large number of guidance and neuroregulatory proteins. The KS proteoglycan microtubule associated protein-1B (MAP1B) stabilizes actin and tubulin cytoskeletal development during neuritogenesis. A second 12 span transmembrane synaptic vesicle associated KS proteoglycan (SV2) provides a smart gel storage matrix for the storage of neurotransmitters. MAP1B and SV2 have prominent roles to play in neuroregulation. Aggrecan and phosphacan have roles in perineuronal net formation and in neuroregulation. A greater understanding of the biology of KS may be insightful as to how neural repair might be improved.

GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain

Keratan sulfate (KS) is a carbohydrate side chain covalently attached to extracellular proteoglycans. KS is composed of disaccharide units of 6-sulfated N-acetylglucosamine (GlcNAc) and galactose. We have previously shown that GlcNAc-6-O-sulfotransferase (GlcNAc6ST) 1 encoded by Chst2 is an enzyme necessary for the synthesis of GlcNAc-6-sulfated KS chains that are required for neuronal plasticity in the visual cortex of the mouse brain during the critical period, but not in adulthood. Here, we show that GlcNAc-6-sulfated KS recognized by the R-10G anti-KS antibody, of which the minimum epitope structure is Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S), distributes diffusely in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net (PNN)-positive neurons in the adult visual cortex. Surprisingly, GlcNAc6ST3, which was discovered as an intestinal GlcNAc6ST encoded by Chst5, is a major brain KS sulfotransferase expressed in oligodendrocytes in adulthood. Moreover, we identified an isoform of the protein-tyrosine phosphatase PTPRZ as a R-10G-reactive KS proteoglycan. These results indicate that GlcNAc6ST3 may play a role in synthesis of a component of PNN in the adult brain, and that the KS-modified isoform of PTPRZ encoded by Ptprz1 could be an extracellular molecule associated with PNNs.

Glycans and glycosaminoglycans in neurobiology: key regulators of neuronal cell function and fate.

The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin sulfate/dermatan sulfate (CS/DS) with selected functional molecules in neural tissues. Cell surface glycans and GAGs have evolved over millions of years to become cellular mediators which regulate fundamental aspects of cellular survival. The glycocalyx, which surrounds all cells, actuates responses to growth factors, cytokines and morphogens at the cellular boundary, silencing or activating downstream signaling pathways and gene expression. In this review, we have focused on interactions mediated by l-fucose, KS and CS/DS in the central and peripheral nervous systems. Fucose makes critical contributions in the area of molecular recognition and information transfer in the blood group substances, cytotoxic immunoglobulins, cell fate-mediated Notch-1 interactions, regulation of selectin-mediated neutrophil extravasation in innate immunity and CD-34-mediated new blood vessel development, and the targeting of neuroprogenitor cells to damaged neural tissue. Fucosylated glycoproteins regulate delivery of synaptic neurotransmitters and neural function. Neural KS proteoglycans (PGs) were examined in terms of cellular regulation and their interactive properties with neuroregulatory molecules. The paradoxical properties of CS/DS isomers decorating matrix and transmembrane PGs and the positive and negative regulatory cues they provide to neurons are also discussed.

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells—primarily eosinophils and mast cells—where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose–acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Immunoproteomic identification of antigenic candidate Campylobacter jejuni and human peripheral nerve proteins involved in Guillain-Barré syndrome

Immunoproteomics is become a potent methodology used for identifying immunoreactive proteins. In this study, an immunoproteomic approach based on 2-dimensional gel electrophoresis (2D-PAGE) and immunoblotting combined with high resolution mass spectrometry (MS) was used to identify immunoreactive proteins that might be involved in mechanisms of Guillain-Barré syndrome (GBS) development, regardless of their potential reciprocal molecular mimicry. Proteins isolated from C. jejuni and human peripheral nerve tissue (HPN) were separated with 2D SDS-PAGE and subjected to western blotting using serum samples from GBS patients. The peptides generated after proteolysis of the immunoreactive proteins were submitted to nanoflow-high performance liquid chromatography-nano electrospray ionization coupled to high resolution mass spectrometry (nHPLC-nESI-MS and MS/MS) followed by SEQUESTdata analysis for proteins identification. In C. jejuni, immunoreactivity was found for GroEL and DnaK, structural proteins (MOMP), key enzymatic proteins necessary for the microbial proliferation (adenylate kinase, enolase, inorganic pyrophosphatase and aspartate ammonia-lyase), and antioxidant enzymes (alkyl hydroperoxide reductase–AhpC and DNA protection during starvation protein - DNA protection factor against Fe2+-mediated oxidative stress).HPN immunoreactive proteins identified were heat shock proteins (HSP), intermediate filaments (vimentin and desmin), and other proteins and enzymes such as troponin/tropomyosin complex and ATP synthase subunit beta and the keratan sulfate proteoglycan lumican. The targeting of vimentin and desmin, suggested that the neuronal autoimmune damage is specifically directed to intermediate neuronal (vimentin) and neuromuscular IF, probably localized nearby cell surface, affording increased accessibility to autoantibodies. These findings suggest that the post-infectious development of GBS may be also associated to additional concomitant immune factors that lead to nerve damage generated by auto-immune trigger(s) different from molecular mimicry.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

The corneal wound healing and the extracellular matrix

The cornea is the first refractive element of the eye. The transparency of the cornea results from the regularly arranged collagen fibrils, forming lamellar structure and the leucin rich proteoglycans, which make interactions between the fibrils. The adult cornea consists mainly of fibril-forming collagens. The cornea has less amount of fibril associated and non-fibrillar collagens. The main proteoglycans of the cornea are keratan-sulfate proteoglycans and it also contains dermatan-sulfate proteoglycans. Disorders of the proteoglycan synthesis lead to the disruption of the unique pattern and result in thicker collagen fibrils. The abnormal structure of the extracellular matrix can generate corneal disorders and the loss of corneal transparency. Furthermore, proteoglycans and collagens have an important role in wound healing. In injury the keratocytes produce higher amounts of collagens and proteoglycans mediated by growth factors. Depending on the ratio of the cells and growth factors the extracellular matrix returns to normal or corneal scar tissue develops.

A Novel KERA Mutation in a Case of Autosomal Recessive Cornea Plana With Primary Angle-Closure Glaucoma.

Keratocan is a cornea-specific keratan sulfate proteoglycan found predominantly in the adult vertebrate eye. In human beings, mutations in keratocan (KERA) are associated with autosomal recessive cornea plana (CNA2), which is characterized by a flattened forward convex curvature of the cornea. Here, we report a novel mutation in a case of autosomal recessive bilateral cornea plana presenting with primary angle-closure glaucoma in a 41-year-old woman from Eastern India. The KERA gene of the patient and her sons was directly sequenced. Mutational analysis of the KERA revealed 2 novel mutations. The first mutation was a 3 base-pair deletion (c.371_373delTCT), leading to the loss of a highly conserved amino acid (p.Phe125del). The second mutation was a base substitution resulting in a silent mutation (c.69G>A). One of her 2 sons carried the homozygous substitution (c.69G>A), whereas the other son was heterozygous (c.69G>R). The mutation that we report here leads to the deletion of a conserved amino acid (p.Phe125del) from the third LRR motif of the keratocan protein, which might lead to an abnormal tertiary structure of the protein, thereby leading to the disease.

Bone sialoprotein keratan sulfate proteoglycan (BSP-KSPG) and FGF-23 are important physiological components of medullary bone

Medullary bone is a specialized bone found in the marrow cavity of laying birds. It provides a significant contribution to the calcium supply for egg shell formation. Medullary bone is distinguished from cortical bone by the presence of large amounts of a keratan sulfate proteoglycan (KSPG). The aims of the present experiment are to confirm the identity of the core protein of KSPG, identify a marker of medullary bone metabolism, and determine whether changes in keratan sulfate (KS) concentration in blood are associated with the egg-laying cycle. Using two different isolation techniques- one specific for bone and another for blood- we have identified bone sialoprotein (BSP) to be the core protein of this KSPG. We also determined that the amount of keratan sulfate (KS) in laying hen blood fluctuates in synchrony with the egg-laying cycle, and thus can serve as a specific marker for medullary bone metabolism. During the course of this investigation, we also found FGF-23 (phosphatonin) to be expressed in medullary bone, in synchrony with the egg-laying cycle. Western blotting was used to demonstrate the presence of this peptide in both laying hen blood and medullary bone extracts. The importance of FGF-23 (phosphatonin) and parathyroid hormone in normalizing the dramatic changes in plasma calcium and phosphorus during the 24h egg-laying cycle is discussed.

Time-dependent localization of high- and low-sulfated keratan sulfates in the song nuclei of developing zebra finches.

Keratan sulfate proteoglycans (KSPGs) and chondroitin sulfate proteoglycans (CSPGs) consist of a protein core with covalently attached glycosaminoglycan side chain. Although CSPGs are known to regulate the end of the critical period, the role of KSPGs in brain development remains unclear. Young male zebra finches memorise song templates during development. The brain regions that are responsible for song learning, known as song nuclei, are recognized as a suitable model for the study of brain development. To understand the potential role of KSPGs, here we examined the localization of KSs with different degrees of sulfation in the brain of developing male zebra finches. Exclusively in the song nuclei, an increase in expression of 5-D-4-positive (5-D-4(+)) high-sulfated KS started after hatching, and reached a plateau at the end of the sensory period, during which the young bird listens to and memorises the song of an adult tutor. By contrast, weak and ubiquitous expression of BCD-4(+) low-sulfated KS remained unchanged until the end of the sensory period, and first increased in the song nuclei at the end of the sensorimotor period, during which the young bird produces plastic songs. Immunoblot analysis showed that phosphacan was a common core protein of 5-D-4(+) KS and BCD-4(+) KS. Finally, we confirmed that the sulfotransferase responsible for the synthesis of high-sulfated KS was exclusively localised in the song nuclei. Our observations suggest that time-dependent localization of KSPGs with different sulfation patterns in the song nuclei may underlie song learning in developing male zebra finches.