A recombinant human LOXL2 secreted from HEK cells displays

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A recombinant human LOXL2 secreted from HEK cells displays complex, acidic glycans at all three N-linked glycosylation sites. Eden P. Go, Hee-Jung Moon, Minae Mure, and Heather Desaire J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00849 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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Journal of Proteome Research

A recombinant human LOXL2 secreted from HEK cells displays complex, acidic glycans at all three N-linked glycosylation sites.

Eden P. Go1, Hee-Jung Moon1, Minae Mure1*, Heather Desaire1*

1

Department of Chemistry, University of Kansas, Lawrence, Kansas, 66047, United

States.

*Correspondence for HD: Phone: 785-864-3015; email: [email protected] *Correspondence for MM: Phone: 785-864-2901; email: [email protected]

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Abstract Human Lysyl oxidase-like 2 (hLOXL2), a glycoprotein implicated in tumor progression and organ fibrosis, is a molecular target for anti-cancer and anti-fibrosis treatment. This glycoprotein contains three predicted N-linked glycosylation sites; one of which is near the protein’s active site and at least one more is known to facilitate the protein’s secretion. Since the glycosylation impacts the protein’s biology, we sought to characterize the native, mammalian glycosylation profile and to determine how closely this profile is recapitulated when the protein is expressed in insect cells. All three glycosylation sites on the protein, expressed in HEK cells, were characterized individually using a mass spectrometry-based glycopeptide analysis workflow. These data were compared to the glycosylation profile of the same protein, expressed in insect cells. We found that the producer cell type imparts a substantial influence on the glycosylation of this important protein. The more relevant version, expressed in HEK cells, contains large, acidic glycoforms; these glycans are not generated in insect cells. The glycosylation differences likely have structural and functional consequences, and these data should be considered when generating protein for functional studies or for high-throughput screening campaigns.

Key words: hLOXL2, glycoprotein, glycosylation, sialic acid, HEK cells, glycopeptide,


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Introduction Glycosylation is an important post-translational modification that impacts about half of all proteins. Glycans can impact the structure,1 binding,2 secretion,3 and circulation half-life4 of proteins, and the glycosylation on a protein can be altered significantly when the same protein is produced under different conditions.5 Specifically, changing the producer cell6 and the construct design7,8 can dramatically impact a protein’s glycosylation, which, in turn, can impact the protein’s properties. Furthermore, the only way to know if the glycosylation profile of any given protein has changed, when it is subjected to new production conditions, is to characterize it. Lysyl oxidase-like 2 (LOXL2) is an example of an important protein whose glycosylation impacts the protein’s biology.9 LOXL2 is a member of lysyl oxidase (LOX)-family of proteins that are copper and lysine tyrosylquinone (LTQ)-dependent amine oxidases.10, 11 LOXL2 catalyzes the oxidation of collagen and elastin to promote crosslinking of these molecules leading to stiffening of the extracellular matrix (ECM). This LOXL2-catalyzed ECM stiffening has been linked to fibrosis and tumor progression.12, 13 Three potential N-linked glycosylation sites are present on this protein, at Asn288, Asn455 and Asn644. The presence, and possibly the type, of its glycans impact the secretion of human LOXL2 (hLOXL2) from HEK298, MCF-7, and MDAMB-231 breast cancer cells.9 Furthermore, the glycans in the amine oxidase catalytic domain at Asn644, likely enhance the stability of the catalytic domain.9 To better understand how glycosylation on this important protein impacts its biology, the glycoforms need to be characterized.



The first analysis of the glycosylation on hLOXL2 was conducted on a truncated form of a recombinant protein lacking the first three of its N-terminal four scavenger receptor cysteine-rich (SRCR) domains secreted from Drosophila S2 cells.14 The analysis showed that all of the appended glycans were relatively small, high-mannose glycoforms, with no charge-baring acidic groups. To date it is yet unknown whether this glycosylation profile is reflective of the native hLOXL2 profile, or whether the insect cell line imparted non-mammalian glycoforms onto the protein. Herein, we identified the glycans on a human cell secreted version of hLOXL2 and compared the results to the glycan profile obtained when this protein is expressed in insect cells. We compared both the type of glycans present at each site and the extent to which each potential glycosylation site was occupied, with the goal of determining whether or not insect cells sufficiently recapitulated mammalian glycosylation. Finally, these studies determined whether the protein carried any glycans that can only be appended in mammalian cells, such as sialic acid or sulfated glycans. This information can be used to determine whether or not expressing hLOXL2 in mammalian cells is necessary in order to obtain a native glycosylation profile, and the studies represent an important step towards understanding how hLOXL2 glycosylation can drive its biological properties.

Experimental Reagents and Materials. Trizma@ hydrochloride, Trizma@ base, urea, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), iodoacetamide (IAM), and glacial acetic acid were purchased from Sigma (St. Louis, MO). Other materials used in 4 ACS Paragon Plus Environment

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this study included Optima™ grade formic acid and Optima™ LC/MS grade acetonitrile (Fisher Scientific), Optima LC/MS grade water (Honeywell Burdick and Johnson), and sequencing grade trypsin (Promega). All reagents and buffers were prepared with deionized water purified to at least 18 MΩ with a Millipore Direct-Q3 Water Purification System. Protein expression and purification. A recombinant hLOXL2 was produced in FreestyleTM HEK suspension culture (Thermo Fisher Scientific) and purified following the method published previously.15 Briefly, FreeStyle™ 293-F cells (HEK cells) in FreeStyleTM 293 Expression Medium were transfected with pcDNA3.1-wt-LOXL2-Strep II (a mammalian expression vector for the wild type-LOXL2 C-terminally fused with a Strep II-tag) using 293fectin™ Transfection Reagent according to the manufacturer’s protocol. After 96 hours of incubation on an orbital shaker (125 rpm) at 37°C with 8% CO2, the culture medium was collected by centrifugation and concentrated using a 30 kDa molecular weight cut-off filter. The WT-LOXL2 was then purified using a gravity flow Strep-Tactin® Sepharose column (IBA). The purity was assessed by 8.5% SDSPAGE analysis and protein concentration was determined by a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Proteolytic Digestion of hLOXL2 Protein. Fifty micrograms of hLOXL2 sample at a concentration of 6.2 mg/mL was denatured with 7 M urea in 100 mM Tris buffer (pH 8.0) and reduced with 5 mM TCEP at room temperature for an hour. Following reduction, the sample was alkylated with 20 mM IAM for another hour in the dark at room temperature. Excess IAM was quenched by adding DTT to a final concentration of 30 mM and incubating for 15 min at room temperature. The reduced and alkylated sample was buffer



exchanged and concentrated using a 30-kDa MWCO filter (Millipore) prior to protease digestion using trypsin at a protein:enzyme ratio of 30:1. After the addition of trypsin, the sample was incubated at 37°C overnight. The reaction was quenched with acetic acid, and the digest was either analyzed immediately or stored at - 20°C until further analysis. To ensure reproducibility of the method, protein digestion was performed at least two times on different days with samples obtained from the same batch and analyzed with using the same experimental procedure. Chromatography and mass spectrometry. LC-MS/MS experiments were performed using an LTQ-Orbitrap Velos Pro (Thermo Scientific) mass spectrometer equipped with ETD (electron transfer dissociation) that was coupled to an Acquity UPLC M-Class (Waters). Five microliters of the digested hLOXL2 sample was separated using a C18 PepMap™ 300 column (300 µm) at a flow rate of 4 µL/min. The mobile phases consisted of solvent A: 99.9% Optima™ LC/MS grade H2O + 0.1% Optima™ grade formic acid and solvent B: 99.9 % Optima™ LC/MS grade CH3CN + 0.1% Optima™ grade formic acid. A CH3CN/H2O multistep gradient was used: 3% B for 5 min, followed a linear increase to 40% B in 50 min, then a linear increase to 90% B in 15 min. The column was held at 97% B for 10 minutes before re-equilibration. A short wash and blank run were performed between each sample run to eliminate any sample carry-over. The following MS and MS/MS parameters were used: data were collected in data-dependent acquisition (DDA) mode, which was set to collect 11 scan events: every high resolution survey scan in the mass range, m/z 400-2000 is followed by 10 MS/MS events. The full MS survey scans were measured at a resolution of 30,000 at m/z 400. Under these conditions, the measured R (FWHM) in the Orbitrap mass analyzer


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at m/z 1000 is 20,000 and 17,000 at m/z 1500. In this study, we employed alternating CID and ETD scans for glycopeptide identification. DDA was set up to acquire 10 scan events consisting of alternating CID and ETD scans after every one full MS scan. A normalized collision energy of 30%, and precursor ion isolation width of 2 Da was used for CID. The ETD precursor ion - radical anion (fluoranthene) reaction was set at an AGC target value of 2x105 and an ion-ion reaction time of 100-150 msec. To improve the ETD efficiency, supplemental activation was turned on at a collisional energy of 25%. The electrospray source was operated in the following conditions: source voltage of 3.0 kV, capillary temperature of 250°C, and S-lens RF value between 50-60%. Glycopeptide identification. Glycopeptide compositions were elucidated using GlycoPep DB16 and GlycoPep ID.17 Details of the compositional analysis have been described previously.7, 8 Briefly, compositional analysis of glycopeptides was carried out by first identifying the peptide portion from tandem MS data. The peptide portion was inferred manually or by Glycopep ID from the Y1 ion, a glycosidic bond cleavage between the two N-acetyl glucosamines at the pentasaccharide core. Once the peptide sequence was determined, plausible glycopeptide compositions were obtained using the high resolution MS data and GlycoPep DB, and the putative glycan candidate was confirmed manually by identifying the Y1 ion and glycosidic cleavages from the CID data. Peptide fragment ions from ETD spectra of glycopeptides identified from a preceding CID scan were manually assessed using Protein Prospector (http://prospector.ucsf.edu). Matched fragment ions within 0.5 Da of the theoretical value were accepted.



Results and Discussion The workflow describing the analytical approach used to fully characterize the glycosylation on hLOXL2 is shown in Figure 1. The protein was reduced, alkylated, and subjected to an in-solution tryptic digestion. The sample was interrogated by LC-MS using data-dependent acquisition, where high-resolution MS data was acquired, along with both CID and ETD data. Glycopeptides were identified in the dataset using a semiautomated approach as described in the experimental section. All glycopeptide assignments were confirmed by high-resolution data, CID data, and ETD data. All assignments were within 10 ppm of the expected masses. Figure 2 shows example MS data containing some of the glycopeptides that were assigned to the Asn644 glycosylation site of hLOXL2. Figure 2A contains a portion of the high-resolution MS data file, where many of the glycopeptides from this site were coeluting. Figures 2B and 2C show CID and ETD data respectively for one of the identified glycopeptides labeled in Figure 2A. Abundant glycosidic cleavage ions are detected in the CID data in Figure 2B, and corresponding peptide fragment ions are detected in the ETD data in Figure 2C further bolstering the assignment. The glycopeptides assigned to the first glycosylation site on hLOXL2, which resides at Asn288 (within the second SRCR domain), are reported in Table 1. At this site, 33 unique glycopeptide compositions are detected. Notably, a nonglycosylated form of the protein is not detected. This experiment confirms that this particular glycosylation site is exclusively occupied with glycans when hLOXL2 is expressed in mammalian cells. Of the numerous glycan types that are present, the majority of them, 19, contain acidic residues. These residues place a negative charge on the glycan under physiological


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conditions, and they can substantially impact the protein’s properties. Sialic acid in particular, which is found on almost half of the glycoforms at this site, is well-known to impact protein clearance rates in vivo.3 The glycans at this site have never been characterized previously, but the data here indicate that this glycosylation profile could not be replicated in non-mammalian producer cells, such as insect cells, because these cell types do not have the glycosylation machinery necessary to append complex glycans. The data in Table 2 summarize the glycosylation profile assigned to the next glycosylation site, Asn455 (within the fourth SRCR domain), and they show that this profile is dramatically different than the profile obtained when the same protein is expressed in insect cells. When the protein was expressed previously in Drosophila S2 cells,14 only a single glycoform was detected; it contained the high mannose core and a single fucose residue. (See Table 2 and reference 14). The peptide baring this glycosylation site is also abundantly detected in its nonglycosylated form when the protein is expressed in insect cells. By contrast, the nonglycosylated form is not detected when the protein is expressed in mammalian cells. Furthermore, the glycans appended to this site are much larger: They contain four to seven residues beyond the trimmanose core. Additionally, all of these glycans are fucosylated, and a third of them are sialylated. The mammalian version of the protein, therefore, could be substantially influenced by both the consistent presence and the large size of the glycans, whereas the glycans would be less likely to impart much influence on the protein when it is expressed in insect cells. Past studies have demonstrated that the glycans at Asn455 are essential for both stability and secretion of the protein.9 When the asparagine at this glycosylation site is mutated to a glutamine, therefore preventing glycosylation from occurring, the protein is no longer



effectively secreted from cells.9 In summary, these data strongly suggest that the mammalian expression system not only provides different glycosylation than the drosophila expression system, it also generates hLOXL2 with a glycosylation profile that more readily promotes protein secretion. The final glycosylation site on hLOXL2, at Asn644 (within the amine oxidase catalytic domain), also contains radically different glycosylation when it is expressed in mammalian cells versus insect cells. The full glycosylation profile of the protein on the mammalian form was assigned as part of this study, and the results are compared, in Table 3, to the glycoforms that were previously characterized when the protein was expressed in insect cells. Use of an insect cell based expression system resulted in hLOXL2 containing small, paucimannose glycans at this glycosylation site. Seven of the nine previously identified glycoforms did not contain an intact high-mannose core.14 By contrast, when the protein is expressed in mammalian cells, 35 different hybrid and complex glycans are obtained, and each of them has the high-mannose core in-tact. In addition, the glycans are almost all fucosylated (80%), and half of them are acidic, baring either sialic acid or sulfate, or both acidic groups. The average size of the glycan differs substantially between the insect-cell expressed protein and the mammalian version. The former has an average of four glycans, while the latter averages eleven. Both the size of the glycan and its charge can contribute to protein-protein interactions by shielding nearby protein epitopes and/or by providing a different local electronic environment; therefore, the differences in glycosylation at this site could bare functional significance. This particular glycosylation site is in the catalytic domain of the protein, and it is nine amino acids away from the LTQ cofactor in the primary sequence, so the glycan types at


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this site may be particularly important if one seeks to identify small molecule inhibitors against hLOXL2. Inhibitors identified based on screens using non-mammalian protein may bind with less affinity against the native, mammalian form of the protein. Since the main outcome of these studies is that the mammalian expression system contains many acidic glycans at each of the glycosylation sites, we additionally wanted to know more about the abundance of these glycans. Were there many acidic glycans, which were each minor components, or did these acidic glycans represent the most abundant species type at each site? To answer this question, we quantified the relative proportion of each glycoform, as described previously;18 and the data can be found in Supplemental Information, Table S1. While this quantitation method does not account for ionization efficiency differences among the different glycoforms,18 it is still useful for answering the question posed here: Are the acidic components the majority species? If all the glycopeptides ionized equally well, the quantity of acidic glycans would be ~70% for the Asn288 site, ~40% for the Asn455 site, and ~60% for the Asn644 site, and since acidic glycopeptides ionize less effectively than neutral glycopeptides (in the positive ion mode),19 these numbers underestimate the proportion of acidic glycans at each site to some degree. In other words, the acidic glycoforms are the majority species. Both the qualitative approach, of counting glycoforms, and the quantitative approach, of considering each glycoform’s relative intensity, come to the same conclusion about the types of glycoforms that are predominant on this protein. Conclusion This study represents the first characterization of the glycosylation profile of hLOXL2, produced in mammalian cells. The glycosylation profile for this form of the



protein is substantially different than the glycosylation profile obtained when the protein is produced in insect cells. Because the glycans have already been implicated in protein secretion, and because at least one glycosylation site is very near the active site of the enzyme, the biological differences reported here likely have functional consequences for the protein. Those conducting further studies of hLOXL2, including the identification of inhibitors, should consider the potential consequences of having mammalian glycosylation present on the protein when choosing a protein expression system.

Acknowledgements: This work was supported by NIH grant R01GM103547 to HD and R01GM113101 and the Kansas Masonic Cancer Research Institute Pilot Research Program of the University of Kansas Cancer Center, P30CA168524 (to M. M.)

Supporting Information: Table S1: All glycoforms detected in this study and their fractional abundances, not accounting for differences in ionization efficiency.


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References 1. Wyss DF, Choi JS, Li J, Knoppers MH, Willis KJ, Arulanandam ARN, Smolyar A, Reinherz EL, Wagner G. Confromation and function of the N-linked glycan in the adhesion domain of Human CD2. Science. 1995, 269, 1273-1278. 2. Wei XP, Decker JM, Wang SY, Hui HX, Kappes JC, Wu XY, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD, Shaw GM. Antibody neutralization and escape by HIV-1. Nature, 2003, 422, 307-312. 3. Chand S, Messina EL, AlSalmi W, Ananthaswamy N, Gao G, Uritskiy G, PadillaSanchez V, Mahalingam M, Peachman KK, Robb ML, Rao M, Rao VB. Glycosylation and oligomeric state of envelope protein might influence HIV-1 virion capture by α4Β7 integrin. Virology 2017, 508, 199-212. 4. Park EL, Mi YL, Unverzagt C, Gabius HJ, Baenziger JU. The asialogllycoprotein receptor clears glycoconjugates terminating with sialic acid alpha 2,6 GalNac. Proc. Nat. Acad. Sci. USA. 2005, 102, 17125-17129. 5. Cox KM, Sterling JD, Regan JT Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nature Biotech. 2006, 24, 1591-1597. 6. Wells EA, Robinson AS. Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement. Biotech. J. 2017, 12, 1600105. 7. Go EP, Ding H, Zhang S, Ringe RP, Nicely N, Hua D, Steinbock RT, Golabek M, Alin J, Alam SM, Cupo A, Haynes BF, Kappes JC, Moore JP, Sodroski JG, Desaire H. Glycosylation benchmark profile for HIV-1 Envelope glycoprotein production based on eleven Env trimers. J. Virol, 2017, 91, VI.02428-16. 8. Go EP, Herschhorn A, Gu C, Castillo-Menendez L, Zhang SJ, Mao YD, Chen HY, Ding HT, Wakefield JK, Hua D, Liao HX, Kappes JC, Sodroski J, Desaire H. Comparative analysis of the glycosylation profiles of membrane-anchored HIV-1 envelope glycoprotein trimers and soluble gp140. J. Virol. 2015, 89, 8245-8257. 9. Xu L, Go EP, Finney J, Moon HJ, Lantz M, Rebecchi K, Desaire H, Mure M. Posttranslational modifications of recombinant human lysyl oxidase-like 2 (rhLOXL2) secreted from Drosophila S2 cells. J. Biol. Chem. 2013, 288, 5357-5363. 10. Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol. 2001, 70, 1-32.



11. Moon HJ, Finney J, Ronnebaum T, Mure M. Human lysyl oxidase-like 2. Bioorg Chem. 2014, 57, 231-241. 12. Barker HE, Cox TR, Erler JT. The rationale for targeting the LOX family in cancer. Nat Rev Cancer, 2012, 12, 540-552. 13. Ikenaga N, Peng ZW, Vaid KA, Liu SB, Yoshida S, Sverdlov DY, Mikels-Vigdal A, Smith V, Schuppan D, Popov YV. Selective targeting of lysyl oxidase-like 2 (LOXL2) suppresses hepatic fibrosis progression and accelerates its reversal. Gut, 2017, 66, 16971708. 14. Rebecchi KR, Go EP, Xu L, Woodin CL, Mure M, Desaire H. A general protease digestion procedure for optimal protein sequence coverage and PTM analysis of recombinant glycoproteins: Application to the characterization of hLOXL2 glycosylation. Anal Chem. 2011, 83(22), 8484-8491. 15. Okada K, Moon H-J, Finney J, Fouture F, Day R, Mure, MM. J.PACE4 proteolytically processes LOXL2 with little impact on its catalytic activity. J. Biol. Chem. 2017. Submitted. 16. Go EP, Rebecchi KR, Dalpathado DS, Bandu ML, Zhang Y, Desaire H. GlycoPep DB: A tool for glycopeptide analysis using a “smart search.” Anal Chem. 2007, 79, 1708-1713. 17. Irungu J, Go EP, Dalpathado DS, Desaire H. Simplification of mass spectral analysis of acidic glycopeptides using GlycoPep ID. Anal Chem. 2007, 79, 3065-3074. 18. Rebecchi KR, Wenke JL, Go EP, Desaire H. Label-free quantitation: A new glycoproteomics approach. J. Am. Soc. Mass Spectrom. 2009, 20(6), 1048-1059. 19. Jiang H, Butnev VY, Bousfield GR, Desaire H. Glycoprotein profiling by electrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 2004, 15, 750-758.


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Figure Legend Figure 1. Workflow depicting the semi-automated analysis used to characterize hLOXL2 glycopeptides.

Figure 2. Example mass spectrometry data used to assign the hLOXL2 glycoforms. (A) High-resolution MS data for a portion of the chromatogram where glycoforms from the N644 site are eluting. (B) CID data for one of the glycopeptides in A. (C) ETD data for one of the glycopeptides in A. The glycopeptide assignments are supported by highresolution MS data, CID, and ETD data.



Figure 1.


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Figure 2.



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Table 1: Glycopeptides identified at Asn288 site Source Peptide Sequence

Glycan

N288VTCENGLPAVVSCVPGQVFSPDGPSR N288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR LGPQVSLDPMKN288VTCENGLPAVVSCVPGQVFSPDGPSR

[Hex]3[HexNAc]4[Fuc]1 [Hex]4[HexNAc]4[Fuc]2 [Hex]3[HexNAc]4[Fuc]2 [Hex]3[HexNAc]5[Fuc]1 [Hex]3[HexNAc]5[Fuc]2 [Hex]3[HexNAc]5[Fuc]1[NeuAc]1 [Hex]3[HexNAc]6[Fuc]1 [Hex]3[HexNAc]6[Fuc]2 [Hex]3[HexNAc]6[Fuc]1[NeuAc]1 [Hex]3[HexNAc]6[Fuc]2[NeuAc]1 [Hex]3[HexNAc]6[Fuc]1[SO3]1 [Hex]3[HexNAc]6[Fuc]2[SO3]1 [Hex]3[HexNAc]6[Fuc]1[SO3]2 [Hex]4[HexNAc]4[Fuc]2 [Hex]4[HexNAc]5[Fuc]1 [Hex]4[HexNAc]5[Fuc]2 [Hex]4[HexNAc]5[Fuc]1[NeuAc]1 [Hex]4[HexNAc]5[Fuc]1[NeuAc]2 [Hex]4[HexNAc]5[Fuc]2[NeuAc]1 [Hex]4[HexNAc]5[Fuc]1[NeuAc]1[SO3]1 [Hex]4[HexNAc]5[Fuc]1[SO3]1 [Hex]4[HexNAc]6[Fuc]1 [Hex]4[HexNAc]6[Fuc]1[SO3]1 [Hex]5[HexNAc]4[Fuc]2 [Hex]5[HexNAc]4[Fuc]1[NeuAc]1[SO3]1 [Hex]5[HexNAc]4[Fuc]1[SO3]1 [Hex]5[HexNAc]5[Fuc]1[NeuAc]1[SO3]1 [Hex]6[HexNAc]6[Fuc]2

a

Note: When this protein was expressed in Drosophila S2 cells, its construct design did not include the second SRCR domain, which contains the Asn288 glycosylation site.


HEK

Drosophila S2 cells a

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Glycan

N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR N455GSLVWGMVCGQNWGIVEAMVVCR

[Hex]3[HexNAc]2[Fuc]1 [Hex]3[HexNAc]5[Fuc]1 [Hex]3[HexNAc]5[Fuc]2 [Hex]3[HexNAc]6[Fuc]2 [Hex]3[HexNAc]6[Fuc]1[[NeuAc]1 [Hex]4[HexNAc]5[Fuc]1 [Hex]4[HexNAc]5[Fuc]2 [Hex]4[HexNAc]5[Fuc]2[NeuAc]1 [Hex]4[HexNAc]5[Fuc]1[NeuAc]1

a

Data from reference 14.


HEK

Drosophila S2 cellsa


Page 20 of 21


Glycan

HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK HYHSMEVFTHYDLLNLN644GTK

[HexNAc]2 [Hex]1[HexNAc]2 [Hex]2[HexNAc]2 [[HexNAc]1[Fuc]1 [[HexNAc]2[Fuc]1 [Hex]1[HexNAc]2[Fuc]1 [Hex]2[HexNAc]2[Fuc]1 [Hex]3[HexNAc]2 [Hex]3[HexNAc]2[Fuc]1 [Hex]3[HexNAc]3[Fuc]1 [Hex]3[HexNAc]4[Fuc]1 [Hex]3[HexNAc]4[Fuc]2 [Hex]3[HexNAc]5[Fuc]1 [Hex]3[HexNAc]5[Fuc]1[NeuAc]1 [Hex]3[HexNAc]5[Fuc]1[SO3]1 [Hex]3[HexNAc]5[Fuc]2 [Hex]3[HexNAc]6[Fuc]1 [Hex]3[HexNAc]6[Fuc]1[NeuAc]1 [Hex]3[HexNAc]6[Fuc]1[SO3]2 [Hex]3[HexNAc]6[Fuc]2 [Hex]3[HexNAc]6[Fuc]2[NeuAc]1 [Hex]3[HexNAc]6[Fuc]2[SO3]1 [Hex]4[HexNAc]4[Fuc]1 [Hex]4[HexNAc]4[Fuc]1[NeuAc]1 [Hex]4[HexNAc]4[Fuc]2 [Hex]4[HexNAc]5[Fuc]1 [Hex]4[HexNAc]5[Fuc]1[NeuAc]1 [Hex]4[HexNAc]5[Fuc]1[NeuAc]1[SO3]1 [Hex]4[HexNAc]5[Fuc]1[SO3]1 [Hex]4[HexNAc]5[Fuc]2 [Hex]4[HexNAc]5[Fuc]2[NeuAc]1 [Hex]4[HexNAc]5[NeuAc]1[SO3]1 [Hex]4[HexNAc]6[Fuc]1[NeuAc]1 [Hex]5[HexNAc]4[Fuc]1 [Hex]5[HexNAc]4[Fuc]1[NeuAc]1 [Hex]5[HexNAc]4[Fuc]2 [Hex]5[HexNAc]5[Fuc]1 [Hex]5[HexNAc]6[Fuc]1

a

Data from reference 14.


HEK

Drosophila S2 cellsa

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For TOC only.


A recombinant human LOXL2 secreted from HEK cells displays

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