key: cord-0262658-sawdvucd
authors: Porell, Ryan N.; Follmar, Julianna L.; Purcell, Sean C.; Timm, Bryce; Laubach, Logan K.; Kozirovskiy, David; Thacker, Bryan E.; Glass, Charles A.; Gordts, Philip L. S. M.; Godula, Kamil
title: Biologically-derived neoproteoglycans for profiling protein-glycosaminoglycan interactions
date: 2022-03-04
journal: bioRxiv
DOI: 10.1101/2022.03.04.482917
sha: 6677a822f71acc4b8add1236126c221acd2291ba
doc_id: 262658
cord_uid: sawdvucd

Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG-microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically-derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells. Graphical Abstract

Proteoglycans (PGs) are abundant on cell surfaces and in the extracellular matrix, where they serve a myriad of biological functions spanning from regulation of growth factor binding and signaling 1,2,3 to tissue development and organ function. 4, 5 They also contribute to pathophysiological processes, including aging and associated disease, 6 immunological responses, 7, 8 and they serve as receptors for infectious agents, such as the Herpes simplex viruses 9 and the SARS-CoV-2 virus, 10 The biological specificity of GAGs is established during their biosynthesis through a nontemplated process via a sequence of enzymatic modifications, which elongate the individual polysaccharide chains and install negatively charged sulfate groups. This results in structurally complex sulfation patterns organized in domains along the polysaccharides that provide highaffinity binding sites for proteins. 11, 12 The structure-function relationships for GAGs remain poorly defined, due to challenges in isolation and structural characterization of GAGs from biological samples, as well as difficulties with producing structurally defined GAG polysaccharides synthetically.

Since their inception in 2002, 13, 14, 15 glycan arrays have become broadly adapted as a high throughput glycomics tool to profile ligand specificity of glycan binding proteins. 16 The glycan array developed by the National Center for Functional Glycomics 17 now contains more than 1,000 unique structures representing N-and O-linked glycans and is considered the gold standard for the field. Parallel pioneering efforts to establish GAG arrays using synthetic HS 18 and CS 19 oligosaccharides provided early insights into GAG structure-function relationships. Advances in chemoenzymatic synthesis of GAG oligosaccharides have significantly accelerated these efforts in recent years. 20, 21, 22 Upwards of 95 synthetic HS structures are now available in an array format, 23 which is still a significant shortfall from the total amount of GAG structures that are found naturally.

Complementing bottom-up synthetic efforts are arrays consisting of biologically-derived GAGs representing the structural complexity of the natural polysaccharides, however, these efforts faced major roadblocks due to difficulties in purification and separation of GAGs into structurally distinct populations. 24 Recent advances in generating glycosylation mutant cell lines through systematic genetic manipulation of GAG biosynthesis are alleviating this limitation by providing access to increased quantities of compositionally-defined bioengineered GAGs. 25, 26 A technical challenge in generating native and bioengineered GAG arrays is the optimization of GAG immobilization onto surfaces. Commonly employed approaches include electrostatic adsorption of the polyanionic glycans onto positively charged poly(lysine)-coated surfaces 27 or precipitation on plastic supports using ammonium chloride. 28 These methods unequally sequester biologically active sulfated domains and influence protein binding. Alternatively, covalent conjugation of GAGs via their reducing ends to amine-, aminooxy-, or hydrazine-functionalized surfaces allows for glycan extension away from the surface, enhancing chain presentation. 29, 30 However, chain grafting efficiency is generally low and varies based on the length and charge of the GAG structure. The inability to characterize the GAGs presentations after immobilization introduces a degree of uncertainty making it difficult to perform comparative analysis of protein binding specificity.

Here, we introduce semi-synthetic neoproteoglycan (neoPG) reagents with a defined molecular architecture to permit comparative analysis of GAG-protein binding interactions. The neoPG has GAG polysaccharides chains end-conjugated to a carrier bovine serum albumin (BSA) protein, improving on existing reductive amination protocols. 31 We employ the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction 32 with a reactive fluorogenic linker to generate the glycoconjugates. The strategy enables efficient coupling with real-time monitoring of GAG conjugation and quantification of the neoPG composition. This novel method is suitable for all members of the GAG family, including tissue-derived and bioengineered polysaccharides and the reagents can be immobilized in an ELISA format to analyze GAG-binding protein interactions or used as soluble reagents to evaluate signaling activity in cells.

Generation of neoProteoglycans (neoPGs). To generate versatile reagents suitable for analysis of GAG-interactions in analytical assays as well as biological assays, we developed a chemical approach for merging polysaccharides with a protein carrier without disrupting ligandbinding domains. Covalent GAG attachment to the protein backbone mimics the organization of native PGs and provides control over GAG valency and presentation both in solution and after immobilization on surfaces. However, macromolecular assemblies of the highly sulfated GAG polysaccharides with other macromolecules, including proteins, necessitates efficient and highyielding bioconjugation chemistries. This is particularly challenging for conjugation of GAGs from biological samples, which can only be isolated in limited amounts or otherwise become costly.

To address these challenges, we have developed a fluorogenic bioorthogonal linker strategy for attaching GAG chains through their reducing ends to a BSA protein carrier. In this process, the GAG chains are furnished with a novel azido-coumarin linker that produces fluorescent light emission (lex/em = 393/477 nm) upon further conjugation with alkynes. This Figure S5 ) on the protein (Figure 2b ). Using this approach, we prepared neoPG conjugates using polysaccharides representing the main classes of GAGs. These included commercially available heparin (Hep, a highly sulfated form of HS), HA, and bovine cartilage CS as well as HS, CS and KS isolated from pig lung and mouse liver tissues (Table S2) . Under optimized conditions, BCN-BSA (~1 nM) was reacted with ACS-GAGs (~10 equiv. per BSA) in PBS buffer at ambient temperature for 20 hrs. Each neoPG was assigned a descriptor GAGx-BSA, where x designates the number of GAG chains per BSA molecule. The conjugation process was efficient, and the maximum number of GAG chains introduced into the neoPGs ranged from x ~ 6-8 for HS, KS and HA and x ~ 8-12 for CS. Both the size and charge of the polysaccharides likely contribute to the overall efficiency of the conjugation process; however, we did not observe any noticeable trends ( Figure 2c and Table S2 ). The composition of the conjugates with respect to GAG chain valency can be tuned by controlling the reagent stoichiometry or reaction time. The neoPG array platform can also be used to analyze the interactions of GAG-modifying enzymes, such as the extracellular human 6-O-endosulfatase 1 (HSulf-1), with their substrates. binding activity; 41, 42, 43, 44 however, the substrate specificity of this enzyme is still poorly defined. 45 First, we tested the effects of HSulf-1 desulfation of immobilized Hep7-BSA on FGF1, FGF2, or VEGFA binding, which were selected based on their differential sensitivity to HSulf-1 activity. 46 We observed significant loss of FGF1 and VEGFA binding but no significant change for FGF2 Relative surface densities of rHS neoPGs after immobilization (100 ng/well) were determined via biotin-azide streptavidin-HRP assay. c) FGF1, FGF2, BMP2, BMP4, and VEGFA binding to rHS neoPGs immobilized at 100 ng/well. d) Ternary complex formation between FGF1 and FGFR1 or 2 in the presence of immobilized Hep7-BSA (100 ng/well) e) Effects of HSulf-1 processing of immobilized Hep7-BSA on FGF2, FGF1, or VEGFA binding (left). Differential processing of rHS neoPGs by HSulf-1 was assessed by FGF1 binding (right). (Bar graphs represent n = 3 replicates, p-values were determined using student's t-test, **p < 0.01, ***p< 0.001, ****p< 0.001,).

structures with GFs observed in binding assays with their biological activities in cells is critical for establishing their structure-activity relationships. We assessed the ability of neoPGs to promote functional pairing between FGF2 and its cell surface receptor FGFR (Figure 5a ). Cultured wild- 

In this report, we outline an efficient and tunable method for generating neoPG conjugates by merging biologically-derived or bioengineered GAG polysaccharides and cyclooctyne-modified BSA protein via a novel bifunctional fluorogenic linker. The method was applicable to all members of the GAG family, including rHS polysaccharides produced in cells with genetically engineered HS biosynthesis. The conjugation process generates a fluorescent signal, which can be used to monitor the progress of the reaction and determine the overall composition of the resulting neoPGs. The reagents were arrayed in 96-well plates to evaluate the ligand specificity of GAGbinding proteins in a convenient ELISA format. Compared to traditional immobilization strategies based on electrostatic adsorption, the conjugation of GAGs to BSA via their reducing-ends provides impartial surface presentation, which enabled analysis of substrate specificity for GAGremodeling lyase and sulfatase enzymes. The neoPGs can also be deployed as soluble probes to confirm their growth factor binding activity in cell signaling assays. With the rapid advancements in precision engineering of cellular glycosylation pathways, these reagents are poised to provide a powerful complement to existing array platforms based on synthetically defined oligosaccharides and contribute a more complete understanding of structure-activity relationships in GAG-protein binding interactions.

To a 4 mL vial containing 3-azido-7-hydroxycoumarin (100 mg, 0.49 mmol, 1.0 eq.) and 4- For enzymatic treatments including heparinase, chondroitinase ABC, hyaluronidase, keratanase II, and 6-O-endosulfatase 1 (HSulf-1), the neoPG immobilized plates were similarly blocked and washed with PBS as above followed by incubation in reaction buffer with or without enzyme present at 37°C for 16 h. Post-enzymatic treatment was followed by three PBS washes and similar protein binding procedures as described above. For Sulf-1 enzyme purification, A375 KDM2B -/cells, kindly provided by Dr. Jeffrey Esko (UCSD), were cultured in OptiMEM for 3-5 days, conditioned media was collected and filtered through a 50 kDa cut-off filter, total protein quantified by BCA assay, and presence of HSulf-1 validated by Western blot analysis and anti-Sulf1 detection. No detectable heparin lyase activity was present in conditioned media.

For biotin-PEG11-azide analyses, BCN-BSA and Hep7-BSA were immobilized onto high-binding 96-well plates in the presence of varying equivalents of the biotin-azide reagent compared to total BCN per BSA (with ~17 BCN/BSA, 0.1 eq = 1.7 biotin-PEG11-azide per BSA and 1 eq = 17 biotin-PEG11-azide per BSA). To quantify neoPG immobilization efficiency, 0.5 eq of biotin-PEG11-azide was utilized in the immobilization assay to account for reacted BCN molecules that were no longer available. Post-immobilization at 4°C for 16 h, wells were washed three times with PBS, blocked with 2% BSA/PBS, and incubated with HRP-conjugated streptavidin for 1.5 h at ambient temperature. Colorimetric analysis of streptavidin binding was conducted similarly to above binding analyses using a kinetic cycle of 370 nm absorbance readings.

Cell surface FGF receptor stimulation assay: FGF2 stimulation and western blotting was performed as detailed previously by the Godula lab. 34 Briefly, mouse embryonic stem cells with endogenous HS production knocked out (Ext1 -/-) were cultured in 6-well plates treated with 0.1% gelatin before being serum starved for 20 h in mESC growth medium lacking FBS. Cells were then treated with 25 ng/mL recombinant human basic FGF (Peprotech) in serum-free medium with heparin, BSA, or Hep7-BSA conjugates (5 µg/mL) for a duration of 15 min at 37°C, 5% CO2.

The cells were then immediately chilled and lysed using a 1X RIPA lysis buffer supplemented with PMSF (1 mM) and 1X protease/phosphatase inhibitor cocktail. Cell lysates were analyzed by BCA assay to determine total protein concentration. 10 µg total protein from each sample was resolved on a 10% SDS-PAGE gel and transferred to a PVDF membrane for blotting. The membrane was blocked with 5% BSA in TBS supplemented with 0.1% Tween-20 (TBST) for a minimum of 1 h at ambient temperature prior to staining (overnight, 4°C) with anti-phospho Erk, anti-total Erk, or anti-alpha tubulin (1:1250, 1:1250, 1:25000 respectively in 5% BSA). After three TBST washes the membrane was incubated with HRP-conjugated secondary antibodies (1:2,000 anti-rabbit HRP and 1:10000 anti-mouse HRP, respectively) for approximately 1.5 h at ambient temperature.

Following a series of TBST washes the blots were visualized using Luminata Forte HRP detection reagent and imaged on a gel scanner (BioRad) for chemiluminescence. For sequential staining, blots were washed in TBST, stripped using Restore PLUS Western blot stripping buffer, washed again in TBST and blocked in 5% BSA for at least 1 h at ambient temperature before further staining. Images were analyzed using ImageJ, with phospho-Erk1/2 and total-Erk1/2 normalized to alpha tubulin, then phospho-Erk was normalized to total-Erk. Lastly, the levels of relative Erk phosphorylation were determined by setting the phosphorylation of Erk in samples containg Ext1 -/-mESCs without FGF2 or neoPG to equal 1.

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Specificity of glycosaminoglycan-protein interactions

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Carbohydrate arrays for the evaluation of protein binding and enzymatic modification

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Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements

Chemoenzymatic Synthesis of Glycosaminoglycans

Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins

Profiling the sulfation specificities of glycosaminoglycan interactions with growth factors and chemotactic proteins using microarrays

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A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships

Microarray method for the rapid detection of glycosaminoglycan-protein interactions

A versatile salt-based method to immobilize glycosaminoglycans and create growth factor gradients

A sulfated glycosaminoglycan array for molecular interactions between glycosaminoglycans and growth factors or anti-glycosaminoglycan antibodies

Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins

Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor

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Stable heparinproducing cell lines derived from the Furth murine mastocytoma

Distinct role of 2-O-, N-, and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1

Minimum structural requirements for BMP-2-binding of heparin oligosaccharides

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

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Sulf Loss Influences N-, 2-O-, and 6-O-Sulfation of Multiple Heparan Sulfate Proteoglycans and Modulates Fibroblast Growth Factor Signaling

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HSulf Sulfatases Catalyze Processive and Oriented 6-O-Desulfation of Heparan Sulfate That Differentially Regulates Fibroblast Growth Factor Activity

Cloning and Characterization of Two Extracellular Heparin-degrading Endosulfatases in Mice and Humans

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We thank Dr Jeffrey D. Esko (UC San Diego) for helpful discussions and for generously providing reagents. This work was supported by the NHLBI K12 Career Development in Glycosciences program K12HL141956 to R.N.P. This work was supported in part by the NIH Director's New Innovator Award (NICHD: 1DP2HD087954-01). KG was supported by the Alfred P. Sloan