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Letters to the Editor  |   February 2014
Determination of Dynamic Changes in the Nature and Biosynthesis of Glycome of wt and rd1 Mice Retinae by Lectin Microarray Analysis
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Investigative Ophthalmology & Visual Science February 2014, Vol.55, 654-657. doi:10.1167/iovs.13-13783
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      Satpal Ahuja; Determination of Dynamic Changes in the Nature and Biosynthesis of Glycome of wt and rd1 Mice Retinae by Lectin Microarray Analysis. Invest. Ophthalmol. Vis. Sci. 2014;55(2):654-657. doi: 10.1167/iovs.13-13783.

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      © ARVO (1962-2015); The Authors (2016-present)

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Understanding the process of retinal glycan biosynthesis and related enzymes, and profiling of the dynamic changes in the retinal glycoproteins is crucial to decode the functions of glycans in retinal pathophysiology. The dynamic changes in mice retinal glycans have been reported previously. 1 In the available retinal Proteomics studies, 2 the glycoproteome was not characterized. Glycans and glycan levels determined by lectin microarray (see fig. 4 from the report of Ahuja 1 ) are categorized below (Sections 1–5) according to the nature of glycan sugars; degree of conversion to di-, tri-, tetra-, oligo-, and polymers; glycan branching; and types of anomeric links (α-, β-, 1→4, 2→3, and so forth) between sugars. This summary is intended to describe the nature of intermediate products, and the role of glycosyl transferase/glycosidase isomers (GT/G) activities involved in glycan biosynthesis/processing during retinal development and degeneration. The categorization of glycans, with respective GT/G, lectins (abbreviated) binding specificity, and significance of differences, presents another facet of the lectin microarray data shown in figure 4 from a prior report by Ahuja. 1 Glycan functions are from the literature. 
GlcNAc Glucosylated and Bisecting GlcNAc Glycans (Glucosyl Transferases/Glucosidases [GT/G])
These include GlcNAc (a monomer, WGA), GlcNAcβ1-4GlcNAc (a di-mer, UDA), GlcNAc tri-mer/tetra-mer, LEL), GlcNAc oligosaccharide (STL), polymer (GlcNAcβ1-4)n (DSA), Complex Type N-glycan with outer Gal, and bisecting GlcNAc (PHA-E). 
In wt and rd1 retinal proteins, GlcNAc glucosylated glycans were the most abundant (46%–48% of the total glycome). The glucosylated glycans were mono-, di-, tri-, tetra-, oligo-, and polymers of GlcNAc glycans linked mainly through β1-4 linkage, and were recognized by the lectins WGA, UDA, LEL, STL, and DSA (see fig. 4 1 for levels of specific lectin ligands). The relative proportion of GlcNAc oligosaccharide glycans in wt and rd1 retinal proteins was highest, and that of GlcNAc monomer was lowest, whereas those of GlcNAc di-, tri-, tetra-, and polymers were intermediate and similar. The level of GlcNAc mono- and oligomers in wt and rd1 did not change with age; GlcNAc dimers increased, but GlcNAc tri-, tetra-, and polymers decreased with age. Biosynthesis of accumulating GlcNAc oligomers and polymers was significantly higher in PN2 and PN14 rd1 retinal proteins compared to those in corresponding wt proteins. 
The relative proportion of Complex Type N-glycan with outer Gal and bisecting GlcNAc glucosylated glycans recognized by the lectin PHA-E (see fig. 4 1 for levels of specific lectin ligands), in wt and rd1 mice retinal proteins, were the least abundant glucosyl glycans. Their level in PN7 rd1 retinal proteins was higher than that in PN2 rd1 proteins. 
During the development and degeneration of wt and rd1 retinae, there was a dynamic increase in the dimerization of GlcNAc to GlcNAcβ1-4GlcNAc and a decrease in that of the (GlcNAcβ1-4)n polymer; however, such changes were less in PN14 rd1 retinal proteins. Relatively lower levels of GlcNAcβ1-4GlcNAc and higher levels of the (GlcNAcβ1-4)n polymer in PN14 rd1 retinal proteins compared to those in PN14 wt proteins suggested an imbalance in the activities of GlcNAc transferase III (GnT-III) isomer for the biosynthesis of glucosylated glycans in rd1 retinae. The GnT-III introduces GlcNAc in β-(1→4) linkage to mannose residues at the base of tri-mannosyl core of the N-glycan to form Complex Type N-glycan with outer bisecting GlcNAc (Figure). An increase in outer bisecting GlcNAc decreases the glycan branching by preventing other isoforms of GTs from accessing the outer bisecting GlcNAc glycans. 35  
Figure
 
Scheme for the biosynthesis of different types of glycans (Fucα1-6GlcNAc, Siaα2-3Galβ1-4GlcNAc, and not Siaα2-6Galβ1-4GlcNAc, Galβ1-4GlcNAc, Manα1-6[Manα1-3]Man, Manα1-6Man, GlcNAcβ1-4GlcNAc, and bisecting GlcNAc) for retinal glycome in mice are from the results of this study, and biosynthesis of Hybrid, bi-, tri-antennary, and bisecting Complex GlcNAc glycans from the precursor High-Man glycan are depicted on this basis. Inner core structure of High-Man N-glycan marked in yellow is common to all these glycans. which are biosynthesized by extension of the High-Man precursor by the relative activities of mannosyl-, glucosyl-, galactosyl-, fucosyl-, and sialyl-transferase, and corresponding glycosidase isomers (GT/G). Drawings of the five glycans are adopted from the brochure “Glycostation,” with permission from GP BioScience, Japan, and now inherited by GlycoTechnica, Ltd. (Yokohama, Japan). Image not available Sia, Image not available Man, Image not available GlcNAc, and Image not available Fuc; Image not availableThick and thin lines between sugars represent β- and α- anomeric linkages.
Figure
 
Scheme for the biosynthesis of different types of glycans (Fucα1-6GlcNAc, Siaα2-3Galβ1-4GlcNAc, and not Siaα2-6Galβ1-4GlcNAc, Galβ1-4GlcNAc, Manα1-6[Manα1-3]Man, Manα1-6Man, GlcNAcβ1-4GlcNAc, and bisecting GlcNAc) for retinal glycome in mice are from the results of this study, and biosynthesis of Hybrid, bi-, tri-antennary, and bisecting Complex GlcNAc glycans from the precursor High-Man glycan are depicted on this basis. Inner core structure of High-Man N-glycan marked in yellow is common to all these glycans. which are biosynthesized by extension of the High-Man precursor by the relative activities of mannosyl-, glucosyl-, galactosyl-, fucosyl-, and sialyl-transferase, and corresponding glycosidase isomers (GT/G). Drawings of the five glycans are adopted from the brochure “Glycostation,” with permission from GP BioScience, Japan, and now inherited by GlycoTechnica, Ltd. (Yokohama, Japan). Image not available Sia, Image not available Man, Image not available GlcNAc, and Image not available Fuc; Image not availableThick and thin lines between sugars represent β- and α- anomeric linkages.
Outer bisecting GlcNAc glycans are associated with growth factor receptors (GFRs) and modify functions of the growth factors. 610 Modified fucosylation and glucosylation of outer bisecting GlcNAc to the GFRs and laminin-5 decreases growth factor signaling, cell migration, and wound healing, and promotes neurodegeneration by inhibiting turnover of GFRs, and the interactions of growth factors with GFRs, and of laminin-5 with galectin-3 through modulation of galectin-lattice structure. 5,1114  
Compared to wt retinal proteins, PN7 rd1 proteins had higher levels of Complex Type N-glycan with outer bisecting GlcNAc, possibly due to an imbalance in the activities of N-glycans processing/branching enzymes. These results suggest that outer bisecting GlcNAc glycans promote degeneration in differentiating rd1 mice retinae by inhibiting laminin-galectin and growth factor signaling. 
Mannosylated Glycans (Mannosyl Transferases/Mannosidases)
These include α-D-Man (LCA), α-D-Man (Calsepa), Man3 core bi-/tri-antennary-Complex Type N-glycan (TxLC-I), Mixtures of Man5 to Man9 (UDA), High-Man, Manα1-3Man (GNA), High-Man, Manα1-6Man (NPA), High-Man, Manα1-6Man, Manα1-3Man (HHL), High-Man, and Manα1-6(Manα1-3)Man (ConA). 
Mannosylated glycans form the second major group (19%–27% of the total glycome) of glycans in the glycome of wt and rd1 retinal proteins, and were α-D-Man, Manα1-3Man, Manα1-6Man, with or without Manα1-3Man branching, Man3 core bi-/tri-antennary-Complex Type N-glycan, mixtures of Man5 to Man9, and High-Man glycans. These glycans were recognized by the lectins LCA, Calsepa, TxLC-I, UDA, GNA, NPA, HHL, and ConA (see fig. 4 1 for levels of specific lectin ligands). The level of Man5 to Man9 glycans was highest and increased with neonatal age, whereas that of High-Man glycans was intermediate. 
Dynamically low and decreasing levels of α-D-Man, high and increasing levels of Man5 to Man9, and consistent intermediate levels of High-Man, both in wt and rd1 retinal proteins, suggest that Man5 to Man9 and High-Man glycans containing Manα1-3Man, Manα1-6Man with or without Manα1-3Man branching are biosynthesized from α-D-Man. High-Man glycans appear to be the precursors for various types of glycans. Addition of GlcNAc to Man5 produces hybrid glycans, which are modified further to Complex glycans by the incorporation of GlcNAc, Gal, Sia, and Fuc through the activities of glucosyl-, galactosyl-, sialyl-, and fucosyl-transferase isomers, respectively (Figure). These diverse isomers of GT associated with the biosynthesis of glycans are generated by modifying the expression of their genes. 3,5,11,12,15 The GT/G in retinal proteins are yet to be quantified and characterized. 
There were similarities in the dynamic levels of mannosylated glycans during retinal development and degeneration in wt and rd1 mice. This may be due to the nature of mannosylated glycans, which act as intermediate precursor for the biosynthesis of Complex glycans. As in mice brain tissue, 16 higher Manα1-6Man, branched Manα1-6(Manα1-3)Man, and Siaα2-3Galβ1-4GlcNAc glycans in wt retinae may bind α-dystroglycan 17 to maintain the integrity of ribbon-synapse-junction, and inhibit myelination of optic nerve head astrocytes, which are constitutively phagocytic in myelination transition zone in wt mice retinae. 16,18 Relatively lower levels of these glycans in PN7, PN14 rd1 proteins and their absence in PN2 rd1 proteins suggest malfunction of the same. 
Galactosylated Glycans (GT/G)
These include GalNAc (TxLC-I), Galβ1-3GalNAc, GalNAc (Jacalin), Galβ1-3GalNAc (ACA), Galβ1-3GalNAc (PNA), α-linked Gal (GSL-IB4), α-linked terminal GalNAc (HPA), Galβ1-4GlcNAc (DSA), and Galβ1-4GlcNAc (RCA-120). 
Galactosylated glycans (19%–22% of the total glycome) of wt and rd1 retinal proteins lacked Galα1-3-GlcNAc. The Galβ1-4GlcNAc glycans form a major component of the galactosylated glycome of retinal proteins recognized by the lectin DSA. The Galβ1-4GlcNAc and α-linked terminal GalNAc, respectively, recognized by the lectins DSA and HPA comprise the main galactosylated glycans, which decreased with age of neonatal mice (see fig. 4 1 for levels of specific lectin ligands). However, in PN14 rd1 retinal proteins the relative proportion of these glycans were higher compared to those in PN14 wt proteins. The glycans GalNAc, Galβ1-3GalNAc, Galβ1-3GalNAc, α-linked terminal GalNAc, and α-linked Gal, respectively, recognized by the lectins Jacalin, ACA, HPA, and GSL-IB4 (see fig. 4 1 for levels of specific lectin ligands), form the second major group of galactosylated glycans in wt and rd1 retinal proteins. The GalNAc, Galβ1-3GalNAc, and Galβ1-4GlcNAc, respectively, recognized by the lectins TxLC-I, PNA, and RCA-120 (see fig. 4 1 for levels of specific lectin ligands), formed a minor component. The proportion of Galβ1-3GalNA and Galβ1-4GlcNAc was significantly higher in PN7 rd1 retinal proteins. 
Age-dependent higher decrease in Galβ1-4GlcNAc and α-linked terminal GalNAc in PN14 wt retinal proteins compared to that in PN14 rd1 proteins suggested an imbalance in the activities of galactosyl transferase and galactosidase isomers during degeneration of rd1 mice retinae. Dynamically higher and lower levels of Complex Type N-glycan with outer Gal, respectively, observed in PN7 rd1 and PN2 rd1 retinal proteins; higher levels of outer Gal, α-linked terminal GalNAc in PN7 rd1, and PN14 rd1 retinal proteins; and of GalNAc, Galβ1-4GlcNAc, and Galβ1-3GalNAc in PN7 rd1 retinal proteins correlate with the retinal degeneration. 19,20 The outer Gal, GalNAc, α-linked terminal GalNAc, Galβ1-4GlcNAc, and Galβ1-3GalNAc galactosylated glycans in retinal proteins are associated mainly with rhodopsin, which is mislocalized and degraded in degenerating rd1, rds mice, and RCS rat retinae (reviewed by Ahuja 1 ). Higher levels of such galactosylated glycans only in PN7 rd1 suggest defective processing of rhodopsin, and an imbalance in the activities of galactosyl transferases and galactosidases. 
Fucosylated Glycans (Fucosyl Transferases/Fucosidases)
These include Fucα1-6GlcNAc(core Fuc) (AOL), Fucα1-6GlcNAc (LCA), Fucα1-6GlcNAc, Fucα1-3(Galβ1-4)GlcNAc (AAL), and Fucα1-2Galβ1-> or GalNAcβ1-> groups at nonreducing end (TJA-II). 
Fucosylated glycans form a minor component (5%–7% of the total glycome) of the wt and rd1 retinal proteins. Fucα1-2-GlcNAc glycans were absent, whereas Fucα1-6GlcNAc(Core Fuc), Fucα1-6GlcNAc, Fucα1-6GlcNAc, and Fucα1-3(Galβ1-4)GlcNAc glycans respectively, recognized by the lectins AOL, LCA, and AAL, constitute the major fucosylated glycans of the retinal proteins (see fig. 4 1 for levels of specific lectin ligands). The proportion of these fucosylated glycans in retinal proteins decreased with age of wt and rd1 mice. The proportion of Fucα1-6GlcNAc and Fucα1-3(Galβ1-4)GlcNAc glycans in PN14 rd1 retinal proteins was significantly higher and that of Fucα1-6GlcNAc(core Fuc) glycans recognized by the lectin AOL were significantly lower in the PN7 rd1 retinal proteins. An increase in Fucα1-6GlcNAc and Fucα1-3(Galβ1-4)GlcNAc glycans, recognized by the lectin AAL, in PN14 rd1 retinal proteins also was significant. The Fucα1-2Galβ1-> or GalNAcβ1-> groups at nonreducing end of the glycome recognized by the lectin TJA-II formed a minor component of fucosylated glycans and were present only in rd1 retinal proteins, especially those from PN7 rd1
Core fucosylation of epidermal growth factor (EGF) receptor, TGF- β1 receptor and α3β1-integrin is required for binding to EGF, TGF-β1, and laminin-5, respectively, to induce Notch, TGF-β1, and laminin functions. 6,7,9,10 Core fucosylation of α3β1-integrin, a major receptor for laminin-5, is involved in cell signaling and migration. 68 Deficiency of the core fucosylation in TGF-β1 receptor increases the activity of MMPs and downregulates pulmonary elastin in mice. 10 Earlier studies showed lower levels of TGF-β1, higher activities of MMPs (reviewed by Ahuja et al. 21 ) and varying degrees of the in vitro rescue effects of lens epithelium-derived growth factor, ciliary neurotrophic factor, brain-derived neurotropic factor, nerve growth factor, and basic fibroblast growth factor (reviewed by Ahuja et al. 22 ) on the photoreceptors of rd1 retinae and support these functions of core fucosylations. 
Sialylated Glycans (Sialyl Transferases/Sialidases)
These include Sia (WGA) and Siaα2-3Galβ1-4GlcNAc (ACG). 
Sialylated glycans form a minor component (4%–7% of the total glycome) of wt and rd1 retinal proteins, which lacked Siaα2-6- glycan. Sia and Siaα2-3Galβ1-4GlcNAc, respectively, recognized by the lectins WGA and ACG, were the only sialylated glycans (see fig. 4 1 for levels of specific lectin ligands), indicating that wt and rd1 mice retinae have limited numbers of active sialyl transferase isomers. The Siaα2-3Galβ1-4GlcNAc glycans were not detected in PN2 wt and rd1 retinal proteins, but their proportions increased with age and were higher in PN14 wt retinal proteins compared to that in PN14 rd1 proteins. Lack of Siaα2-3Galβ1-4GlcNAc in PN2 wt and rd1 retinal proteins indicated absence of Siaα2-3 sialyl transferases and ribbon-synapse-junctions at PN2 stage. 
Balance between the activities of sialyl-transferases and sialidases maintains the level of sialylation in the inner segments of rod photoreceptors. Sialo glycans with terminal Sia are associated with rod photoreceptors and the interphotoreceptor matrix. 20  
Sialyl-transferase isomers–mediated sialylation has a crucial role in cell adhesion and migration, 23 and may be important for the normal development of mice retinae. Sialyl-transferase (ST3Gal IV) incorporates Siaα2-3 into terminal Galβ1-4GlcNAc, Galβ1-3GalNAc structures of glycoproteins, namely α-dystroglycan, and maintains electrophysiological integrity of ribbon-synapse-junctions in wt mice retinae. 24 The relatively higher biosynthesis of Siaα2-3Galβ1-4GlcNAc glycan in wt retinal proteins may be used as a marker for the integrity of ribbon-synapse-junctions. 
Multi lectins reacting with glycoproteins show better avidity of binding with glycan ligands. 25 Five groups of lectins mentioned above reacted with retinal proteins glycome, and each group of lectins may be used to enrich/fractionate retinal glycoproteins into five corresponding groups, respectively, having glucosylated-, mannosylated-, galactosylated-, fucosylated-, and sialylated-glycans before characterization by glycoproteomics, and activities of GT/G may be measured by enzyme-linked lectin-ligand assay. 
Conclusions
Decrease in core fucosylation, and increase in outer bisecting GlcNAc glucosylation and galactosylation of rd1 retinal proteins may be used as marker for retinal degeneration. Dynamic differences in the biosynthesis of different types of glycans indicated a variation/imbalance in the activities of GT/G during the retinal development and degeneration. Therefore, determination of the levels of glycosyl transferases may help in understanding the biosynthesis of the glycan, and inhibition of glucosyl- and galactosyl-transferases may slow the progress of photoreceptor degeneration. 
Acknowledgments
The author thanks Sten Andréasson for infrastructural facilities; Masao Yamada, Kyoko Yokota, and Ryoko Sawada (GP BioSciences Ltd., Yokohama, Japan) for lectin microarray analyses; Birgitta Klefbohm for collecting retinae; Per Ekström for providing mice; and Poonam AhujaJensen and Sanjay Ahuja for statistical analyses. 
Supported by Ögonfonden Synfrämjande Forskning, Stöd ögonforskningen, Umeå; Stiftelsen Kronprinsessan Margaretas Arbetsnämnd för synskadade (KMA); and Stiftelsen för synskadade i.f.d. Malmöhus Län, (Sweden). 
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Figure
 
Scheme for the biosynthesis of different types of glycans (Fucα1-6GlcNAc, Siaα2-3Galβ1-4GlcNAc, and not Siaα2-6Galβ1-4GlcNAc, Galβ1-4GlcNAc, Manα1-6[Manα1-3]Man, Manα1-6Man, GlcNAcβ1-4GlcNAc, and bisecting GlcNAc) for retinal glycome in mice are from the results of this study, and biosynthesis of Hybrid, bi-, tri-antennary, and bisecting Complex GlcNAc glycans from the precursor High-Man glycan are depicted on this basis. Inner core structure of High-Man N-glycan marked in yellow is common to all these glycans. which are biosynthesized by extension of the High-Man precursor by the relative activities of mannosyl-, glucosyl-, galactosyl-, fucosyl-, and sialyl-transferase, and corresponding glycosidase isomers (GT/G). Drawings of the five glycans are adopted from the brochure “Glycostation,” with permission from GP BioScience, Japan, and now inherited by GlycoTechnica, Ltd. (Yokohama, Japan). Image not available Sia, Image not available Man, Image not available GlcNAc, and Image not available Fuc; Image not availableThick and thin lines between sugars represent β- and α- anomeric linkages.
Figure
 
Scheme for the biosynthesis of different types of glycans (Fucα1-6GlcNAc, Siaα2-3Galβ1-4GlcNAc, and not Siaα2-6Galβ1-4GlcNAc, Galβ1-4GlcNAc, Manα1-6[Manα1-3]Man, Manα1-6Man, GlcNAcβ1-4GlcNAc, and bisecting GlcNAc) for retinal glycome in mice are from the results of this study, and biosynthesis of Hybrid, bi-, tri-antennary, and bisecting Complex GlcNAc glycans from the precursor High-Man glycan are depicted on this basis. Inner core structure of High-Man N-glycan marked in yellow is common to all these glycans. which are biosynthesized by extension of the High-Man precursor by the relative activities of mannosyl-, glucosyl-, galactosyl-, fucosyl-, and sialyl-transferase, and corresponding glycosidase isomers (GT/G). Drawings of the five glycans are adopted from the brochure “Glycostation,” with permission from GP BioScience, Japan, and now inherited by GlycoTechnica, Ltd. (Yokohama, Japan). Image not available Sia, Image not available Man, Image not available GlcNAc, and Image not available Fuc; Image not availableThick and thin lines between sugars represent β- and α- anomeric linkages.
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