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. 2022 Oct 25;5(11):1062–1069. doi: 10.1021/acsptsci.2c00153

Disrupting N-Glycosylation Using Type I Mannosidase Inhibitors Alters B-Cell Receptor Signaling

Aric Huang †, Suresh E Kurhade ‡, Patrick Ross ‡, Kyle D Apley †, Jonathan Daniel Griffin §, Cory J Berkland †,§,∥, Mark P Farrell ‡,*
PMCID: PMC9667535  PMID: 36407961

Abstract

graphic file with name pt2c00153_0004.jpg

Kifunensine is a known inhibitor of type I α-mannosidase enzymes and has been shown to have therapeutic potential for a variety of diseases and application in the expression of high-mannose N-glycan bearing glycoproteins; however, the compound’s hydrophilic nature limits its efficacy. We previously synthesized two hydrophobic acylated derivatives of kifunensine, namely, JDW-II-004 and JDW-II-010, and found that these compounds were over 75-fold more potent than kifunensine. Here we explored the effects of these compounds on different mice and human B cells, and we demonstrate that they affected the cells in a similar fashion to kifunensine, further demonstrating their functional equivalence to kifunensine in assays utilizing primary cells. Specifically, a dose-dependent increase in the formation of high-mannose N-glycans decorated glycoproteins were observed upon treatment with kifunensine, JDW-II-004, and JDW-II-010, but greater potency was observed with the acylated derivatives. Treatment with kifunensine or the acylated derivatives also resulted in impaired B-cell receptor (BCR) signaling of the primary mouse B cells; however, primary human B cells treated with kifunensine or JDW-II-004 did not affect BCR signaling, while a modest increase in BCR signaling was observed upon treatment with JDW-010. Nevertheless, these findings demonstrate that the hydrophobic acylated derivatives of kifunensine can help overcome the mass-transfer limitations of the parent compound, and they may have applications for the treatment of ERAD-related diseases or prove to be more cost-effective alternatives for the generation and production of high-mannose N-glycan bearing glycoproteins.

Keywords: B-cell receptor, glycoscience, kifunensine, high-mannose N-glycans, mannosidase inhibitors, primary B cells

N-Glycosylation is a post-translational modification where oligosaccharides are covalently attached to asparagine residues. This process begins in the endoplasmic reticulum (ER) with the transfer of a precursor oligosaccharide, followed by subsequent processing (i.e., addition and removal of sugar residues) mediated by a variety of enzymes as the protein moves from the ER to the Golgi apparatus (Figure 1A). Importantly, glycan structure can alter a protein’s biological function by affecting protein stability, structure, and function.1,2 Alterations to N-linked glycosylation have been attributed to various diseases such as autoimmune diseases,3,4 congenital disorders,5 and cancers.6,7

Figure 1.

Figure 1

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Biosynthesis of N-linked glycans and small-molecule inhibitors of mannosidase I. (A) N-linked glycosylation begins in the endoplasmic reticulum (ER), where a 14-sugar precursor is transferred to an Asn residue of a protein by the enzyme, oligosaccharyltransferase (OST). Glucose residues are cleaved by α-glucosidase I and II (Glc I and Glc II), forming high-mannose N-glycans. A mannose residue is then removed by mannosidase I (Man I) in the ER. Glycoproteins are moved to the Golgi apparatus for further glycan modifications. At the Golgi mannose residues are removed by Man I and mannosidase II (Man II), and a series of transferases (e.g., N-acetylglucosaminetransferase I and II (GnT I and II), galactosyltransferase (GalT), and sialyltransferase (ST)) catalyze the formation of complex N-glycans. (B) Chemical structures of compounds used in the study and their corresponding calculated partition coefficient (CLogP) values. More hydrophobic compounds have higher cLogP values. cLogP values were calculated using ChemDraw v20.1.1.

Kifunensine is a selective type I α-mannosidase (Man I) inhibitor (Figure 1). Specifically, it has been shown to inhibit endoplasmic reticulum and Golgi localized Man I enzymes.8 Therefore, kifunensine mediated disruption of the N-glycosylation biosynthetic pathway results in an increased prevalence of high-mannose N-glycans and a reduction in the formation of hybrid- and complex-type N-glycans.9,10 Kifunensine has also been reported to have therapeutic potential for the treatment of ERAD-related diseases8,11 (e.g., Alzheimer’s disease,12 lysosomal storage disorders,13 catecholaminergic polymorphic ventricular tachycardia,14 sarcoglycanopathies15,16), cancer,17−19 viral infections,20−22 and it is also applied for the preparation of high-mannose N-glycan bearing glycoprotein therapeutics.9,10,23−25 However, the widespread use of kifunensine is limited by its high hydrophilicity, which hampers its ability to permeate tissue and enter cells (i.e., mass-transfer limitations).19,26−28 We have previously described the preparation of acylated derivatives of kifunensine (i.e., JDW-II-004 and JDW-II-010) that exhibit >75-fold greater potency than kifunensine in assays leveraging immortalized cells.27

In this study, the activity of the hydrophobic kifunensine derivatives in primary mouse splenocytes is determined by assessing the induction of high-mannose N-glycans. Additionally, the impact of Man I inhibition, using kifunensine and the kifunensine derivatives, in B-cell receptor signaling in both mouse and human B cells is determined.

Results

Kifunensine and the Acylated Derivatives of Kifunensine Promoted the Formation of High-Mannose N-Glycans in Primary Mouse B Cells

The previously described hydrophobic acylated derivatives of kifunensine may overcome the mass-transfer limitations of the parent compound, kifunensine (Figure 1B).27 The kifunensine analogue SK-III-122 is used as a negative control for this study, as our preliminary findings indicate that SK-III-122 failed to increase the prevalence of high-mannose N-glycans. The activity of the acylated kifunensine derivatives in primary mouse splenocytes derived from NOD mice was initially determined. Primary mouse splenocytes were treated with different concentrations of kifunensine, JDW-III-004, and JDW-II-010 for 48 h. The cells were then stained with cell-specific markers and the high mannose-specific lectin cyanovirin-N and subsequently analyzed by flow cytometry. We observed a dose-dependent increase in high-mannose N-glycan formation in the splenocyte cells (Figure S1A). Further flow cytometric analysis demonstrated that the increase in high-mannose glycans was predominately attributed to CD19+ B cells, and not to CD3+ T cells (Figure S1B and S1C). Moreover, the hydrophobic acylated derivatives of kifunensine increased the prevalence of high mannose N-glycans on CD19+ B cells more efficiently than kifunensine. We also investigated if the treatment of splenocytes with kifunensine and the kifunensine derivatives altered CD20 or CD22 levels, as the upregulation of these B-cell surface markers may potentiate the efficacy of anti-CD20 or anti-CD22 antibody therapies.29,30 However, the treatments did not significantly alter CD20 or CD22 levels on the B cells (Figure S1D,E).

Kifunensine and the Acylated Derivatives of Kifunensine Impaired BCR Signaling, Increased CD79b Levels, and Decreased CD19 Levels in Mouse B Cells

Next, we explored if kifunensine and the kifunensine derivatives altered B-cell receptor (BCR) signaling. Preliminary Ca2+ flux experiments to assess BCR signaling was performed under similar conditions to the above study; however, the primary splenocyte cells did not respond well to Ca2+ flux monitoring after 48 h. Therefore, subsequent studies were performed with a shorter 24-h treatment period to better monitor Ca2+ flux, and a high treatment concentration (i.e., 100 μM) was used to better observe any changes due to the treatments. Specifically, fresh splenocytes were isolated from NOD mice, and the cells were treated with 100 μM kifunensine or derivatives for 24 h. Splenocytes were stained with the pan B-cell marker, B220, to identify the B-cell population by flow cytometry. To assess changes in BCR signaling activity, the cells were treated with anti-IgM F(ab’)2, and the change in Ca2+ flux was monitored by flow cytometry. Treatments resulted in reduced anti-IgM F(ab’)2 induced Ca2+ flux (Figure 2A). Treatments did not affect the surface levels of IgM (Figure 2B); however, increased surface levels of the BCR signaling component Igβ (CD79b, Figure 2C) and decreased surface levels of the coreceptor CD19 (Figure 2D) were observed. To assess the changes in glycosylation, the treated cells were stained with a lectin specific for high mannose N-glycans (i.e., CVN), α2,3-linked sialic acids (i.e., MALII), or α2,6-linked sialic acids (i.e., SNA).31 The inhibition of Man I resulted in elevated levels of high mannose N-glycans (Figure 2E) and glycans with α2,3-linked sialic acids (Figure 2F), but decreased levels of glycans with α2,6-linked sialic acids were observed (Figure 2G). No effects were observed when the cells were treated with the negative-control SK-III-122.

Figure 2.

Figure 2

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Kifunensine treatment inhibited B-cell receptor signaling in mice B220+ B cells via decreasing surface CD19 levels. Fresh splenocytes were isolated from NOD mice, plated in 24-well plates at 1.5 × 106 cells/0.5 mL, and were treated with 100 μM kifunensine, JDW-II-004, JDW-II-010, or SK-III-122 (in 0.05% DMSO) for 24 h. Splenocytes were stained with anti-B220 for flow cytometric analysis of the B-cell population. (A) Changes in calcium flux of B220+ B cells were assessed by staining splenocytes with the calcium indicator, Fluo-4 AM, and performing flow cytometric analysis upon B-cell receptor cross-linking with anti-IgM F(ab’)2 over 3 min. Arrow on the x-axis indicates the time of anti-IgM F(ab’)2 addition. Calcium flux curves are plotted as the average MFI readings. Flow cytometry was also used to measure the surface expression of (B) IgM, (C) CD79b, and (D) CD19. The cells were also stained with lectins that recognize (E) high-mannose oligosaccharides (CVN), (F) α2,3-linked sialic acids (MALII), and (G) α2,6-linked sialic acids (SNA), and the binding of lectins was determined by flow cytometry. MFI levels are normalized to DMSO-treated controls. Data are plotted as mean with SD. One-way ANOVA with Dunnett’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs DMSO group, n = 3. Data are representative of three independent experiments.

Kifunensine and the Acylated Derivatives of Kifunensine Facilitated BCR Signaling and Increased BCR Levels but Decreased CD19 Levels in Human B Cells

Next, we investigated whether the changes in BCR signaling induced by kifunensine and the acylated derivatives are translatable to human B cells. Primary human peripheral blood mononuclear cells (PBMCs) were treated with kifunensine or the kifunensine derivatives for 24 h and changes in calcium flux and BCR signaling molecules were assessed by flow cytometry. Interestingly, calcium flux experiments showed that JDW-II-010 modestly increased BCR signaling in primary human B220+ B cells (Figure 3A); an opposite effect from that observed with the mouse B cells (Figure 2A). Treatment with kifunensine and the derivatives did not affect IgM levels (Figure 3B), but kifunensine and the acylated derivatives increased CD79b levels (Figure 3C), while a significant decrease in CD19 levels was only observed with JDW-II-010. Furthermore, lectin staining showed that the kifunensine and the acylated derivatives of kifunensine successfully increased high-mannose N-glycans levels (Figure 3E) and decreased α2,6-linked sialic acid glycans (Figure 3G), but interestingly, no significant changes in α2,3-linked sialic acid glycans levels were observed (Figure 3F).

Figure 3.

Figure 3

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Effects of kifunensine and the acylated derivatives of kifunensine on B cells of primary human PBMCs. Fresh human PBMCs were plated in 24-well plates at 1.5 × 106 cells/0.5 mL and treated with 100 μM kifunensine, JDW-II-004, JDW-II-010, or SK-III-122 (in 0.05% DMSO) for 24 h. Cells were stained with anti-B220 for analysis of the B-cell population. (A) Changes in calcium flux of B220+ B cells were assessed by staining splenocytes with the calcium indicator, Fluo-4 AM, and performing flow cytometric analysis upon B-cell receptor cross-linking with anti-IgM F(ab’)2 over 3 min. Arrow on the x-axis indicates the time of anti-IgM F(ab’)2 addition. Calcium flux curves are plotted as the average MFI readings. Flow cytometry was used to measure the surface expression of (B) IgM, (C) CD79b, and (D) CD19. The cells were also stained with lectins that recognize (E) high-mannose oligosaccharides (CVN), (F) α2,3-linked sialic acids (MALII), and (G) α2,6-linked sialic acids (SNA) and the binding of lectins was determined by flow cytometry. MFI levels are normalized to DMSO-treated controls. One-way ANOVA with Dunnett’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs DMSO group, n = 3. Data are plotted as mean with SD. Data are representative of two independent experiments.

We also investigated the effects of the treatments on Raji B cells, an immortalized human B-cell line. Raji B cells that were treated with the acylated derivatives of kifunensine (i.e., JDW-II-004 and JDW-II-010) had a slightly increased BCR signaling compared to the cells treated with DMSO vehicle and control compound SK-III-122, but differences in BCR signaling were not statistically significant (Figure S2A). Treating the cells with kifunensine and the acylated derivatives also increased surface levels of BCR (i.e., IgM and CD79b, Figure S2B,C), but CD19 levels were only decreased upon JDW-II-004 and JDW-II-010 treatment (Figure S2D). Lectin staining demonstrated that kifunensine and the acylated derivatives increased the levels of high mannose N-glycans (Figure S2E), but relatively minimal alterations in the levels of sialic acid-containing glycans were observed (Figure S2F,G).

Discussion

We previously reported kifunensine and the acylated derivatives, JDW-II-004 and JDW-II-010, as effective inhibitors of type I mannosidase enzymes using immortalized tumorigenic human B cells (Raji B), T cells (Jurkat), and epithelial cells (MDA-MB-231).27 Here, the activity of the compounds was assessed using more biologically relevant primary cells.

In primary mouse B cells, we observed that kifunensine and the acylated derivatives of kifunensine inhibited BCR activation signaling, as evidenced by a reduction in calcium flux upon BCR cross-linking. Interestingly, this correlated with the decreased cell surface levels of CD19. Since we did not see changes in CD22 levels the reduction in BCR activation is not likely due to the inhibitory effects of this receptor. Our results are similar to the findings of Mortales et al. (2020), in which they demonstrate that primary mouse B cells deficient in GnT I (i.e., the enzyme involved in the next step of N-glycan biosynthesis after Man I, see Figure 1A) displayed decreased BCR signaling, which they attributed to lower surface IgM and CD19 levels.32 They determined that the lower levels of CD19 resulted from a higher rate of endocytosis, leading to its degradation. Using CD22-knockout studies, Mortales et al. also demonstrated that the decreased BCR signaling was independent of the inhibitory receptor CD22. In our study, the inhibition of Man I in mouse B cells also displayed decreased BCR signaling that may be due to lower CD19 levels, and independent of CD22. However, we did not observe decreases in BCR levels (i.e., IgM and CD79b). Indeed, our studies and the studies conducted by Mortales et al. used different mice (i.e., NOD vs C57BL/6 mice), different inhibitory methods (i.e., pharmacological inhibition vs gene knockout), and target different enzymes (i.e., Man I vs GnT I) and therefore would generate different types of N-glycans. Nevertheless, both studies target the early stages of N-glycan biosynthesis and it was interesting to see that we observe similar results, demonstrating the importance of complex N-glycans for B-cell function.

Through lectin staining, we observed that kifunensine and the acylated derivatives of kifunensine increased the levels of high-mannose N-glycans and decreased the levels of α2,6-linked sialic acid containing glycans in mouse and human B cells. The former observation was not surprising given the Man I inhibitory properties of these compounds. Interestingly, kifunensine and the acylated derivatives increased the levels of glycans with α2,3-linked sialic acids in mouse B cells, but this was not observed in human B cells. We reason that the increased prevalence of α2,3-linked sialic acids may be due to the increased sialylation of O-glycans and glycolipids, as the inhibition of Man I enzymes should effectively reduce the levels of N-glycans that can be decorated by sialyltransferase enzymes.4 It is also possible that inhibiting the formation of complex N-glycans results in the accumulation of the sialyltransferase substrate CMP-sialic acid, which could be used by α2,3-sialyltransferase enzyme to sialylate O-glycans and glycolipids. The disparity between the changes in sialylation observed in mouse and human derived B cells requires further investigation at this time but it is important to note that glycosylation patterns can vary significantly depending on cell type and species.33

As demonstrated by the increased prevalence of high-mannose glycans on primary mouse and human B cells and Raji B cells, kifunensine and the acylated derivatives successfully inhibit Man I in both mouse and human B cells. We also found that inhibiting Man I resulted in an elevation of CD79b levels (a component of the BCR) and a reduction in the levels of surface CD19, a major coreceptor for BCR signal transduction. Despite the consistent effects of Man I inhibition, we also found differences in response with the different cell types tested. For instance, Man I inhibition increased surface IgM levels in the immortalized human B-cell line (i.e., Raji B cells), but this was unaltered in primary mouse and human B cells. In addition, Man I inhibition decreased BCR-induced calcium flux in the mouse B cells. Interestingly, the magnitude of these changes in BCR signaling appeared to correlate with the decrease in CD19 levels.

Since CD19 is considered a costimulatory protein that lowers the threshold for B-cell activation and amplifies BCR signaling,34,35 it was not surprising that a decrease in CD19 levels was correlated with a decrease in calcium flux in the mouse B cells. Interestingly, a decrease in CD19 level was correlated with an increase in calcium flux in human B cells, which may suggest a B-cell inhibitory effect of CD19. However, there have been previous reports of CD19 in activating or inhibiting B-cell responses depending on the stimulating conditions and the degree of CD19 cross-linking.36−39 In addition, the differences observed depending on the source of the B cells may highlight the differences in cell biology, such as interspecies differences in cell biology40,41 or glycan formation.42

A limitation of comparing mouse and human B cells also arises from differences in cell surface markers. Here, we used anti-CD45R/B220 antibodies to select for the B-cell populations and used anti-IgM F(ab’)2 fragment for cross-linking BCRs. Although B220 can be used as a pan B-cell marker for mice, it is not a pan B-cell marker for human.43,44 This is because CD45R expression on human B cells is dynamic and is downregulated during germinal center differentiation into memory/plasma B cells.45 Nevertheless, the anti-CD45R/B220 antibody should capture the majority of the human B cells found in PBMCs,44,46 and the Ig class-switched memory-B cells that would not have been cross-linked by the anti-IgM F(ab’)2 fragment.

Conclusions

Together, these studies demonstrate the effectiveness of kifunensine and the acylated derivatives of kifunensine in inhibiting Man I and promoting high mannose N-glycan formation in primary cells. However, considerations of relevant cell types are important depending on the applications of the studies as the biological effects of Man I inhibition can vary depending on the type of cells used. The acylated derivatives of kifunensine we used in this study were functionally equivalent to kifunensine, but their higher potency, mediated by their higher hydrophobicity, may help overcome the mass-transfer limitation of kifunensine.19,26−28 Therefore, these kifunensine derivatives will be useful in supporting research investigating the therapeutic potential of inhibiting Man I, as well as cost-effective alternatives for generating high mannose glycoprotein therapeutics.26

Materials and Methods

Kifunensine and Synthesis of Kifunensine Derivatives

Kifunensine, JDW-II-004, and JDW-II-010 were synthesized as previously described.27,47 A reaction scheme for the synthesis of kifunensine, JDW-II-004, and JDW-II-010 is shown in Figure S3. Briefly, kifunensine diacetonide was reacted with 75% TFA to generate kifunensine.47 Kifunensine diacetonide (800 mg, 2.561 mmol) was also reacted with p-methoxybenzyl chloride (PMB-Cl, 510 μL, 3.823 mmol) in the presence of K2CO3 (700 mg, 5.102 mmol) and tetrabutylammonium iodide (39 mg, 0.256 mmol) in anhydrous acetone (20 mL) at 60 °C for 5 h.27 The reaction product was then purified by filtration, concentrated, dissolved in water, extracted with EtOAc, washed with brine, desiccated over Na2SO4, and further purified by CombiFlash chromatography using EtOAc in hexanes (1:1). The product was then dissolved in MeOH and reacted with concentrated HCl at room temperature for 18 h. The reaction product was purified by CombiFlash chromatography using MeOH in DCM (9:1), generating N-PMB-kifunensine. N-PMB-kifunensine was then acylated by reacting with propionic or butanoic anhydride (for generation of JDW-II-004 or JDW-II-010, respectively) in anhydrous pyridine and 4-dimethylaminopyridine (DMAP) for 18 h at room temperature. The reaction was then diluted with water, extracted with EtOAc, washed with HCl, washed with brine, dried over Na2SO4, concentrated, and further purified by CombiFlash chromatography using EtOAc in hexanes (1:1). The acylated product then underwent PMB deprotection by the addition of ceric ammonium nitrate (CAN) in ice-cold 1:1 acetonitrile–water mixture. The reaction was warmed to room temperature and reacted for another 1.5 h. The reaction was then concentrated, diluted with water, extracted with EtOAc, washed with brine desiccated over Na2SO4, filtered, concentrated, and purified by CombiFlash chromatography using EtOAc in hexanes (1:1) to obtain JDW-II-004 and JDW-II-010.

The synthesis of control compound SK-III-122 will be described elsewhere in due course. Analytical data for SK-III-122 is as follows: 1H NMR (500 MHz, methanol-d4) δ 7.45–7.36 (m, 4H), 7.36–7.31 (m, 1H), 4.77 (d, J = 9.6 Hz, 1H), 4.44 (dd, J = 8.2, 4.5 Hz, 1H), 4.24 (t, J = 7.0 Hz, 1H), 4.09–4.04 (m, 1H), 4.01 (t, J = 3.4 Hz, 1H), 3.93 (dt, J = 9.7, 1.9 Hz, 1H), 3.72 (ddd, J = 11.6, 7.6, 1.0 Hz, 1H), 3.65 (ddd, J = 11.5, 6.3, 1.0 Hz, 1H), 3.42 (dd, J = 15.1, 4.5 Hz, 1H), 3.21 (dd, J = 15.0, 8.2 Hz, 1H) (Figure S4). 13C NMR (126 MHz, methanol-d4) δ 167.8, 134.5, 129.0, 128.7, 128.6, 127.4, 71.9, 68.9, 68.5, 66.8, 59.5, 58.8, 58.7, 34.8 (Figure S5). HRMS: calcd [M + H]+ 309.1450 m/z, found 309.144 m/z.

Cell Culture

All cells used in this study were maintained in complete Roswell Park Memorial Institute (cRPMI) medium, which was prepared by combining RPMI Medium 1640 (Gibco no. 11875, Paisley, United Kingdom) with 10% fetal bovine serum and 1% penicillin streptomycin. The cells were plated in 24-well plates at the indicated concentrations and cultured in an incubator maintained at 37 °C, 5% CO2. Raji B cells were obtained from American Type Culture Collection (no. CCL-86, Manassas, VA). Human PBMCs were purchased from StemCell (no. 200-0077 Kent, WA).

Mouse splenocytes were isolated from NOD/ShiLtJ mice (Jackson Laboratory no. 001976, Bar Harbor, ME) and put into a solution of ice-cold DPBS (Gibco no. 14190, Paisley, United Kingdom). No experimental differences were observed between splenocytes isolated from male or female mice. Spleen samples were dissociated by mechanical digestion by using the end of a 1 mL syringe plunger to pass splenocytes through a sterile wire mesh. The dissociated splenocytes were then centrifuged at 200g for 5 min at 4 °C, resuspended with 1 mL of red cell lysing buffer Hybri-Max (Sigma no. R7757, St. Louis, MO), incubated for 7 min at room temperature, and 9 mL of cRPMI medium was added to stop the lysis buffer. The cells were centrifuged again resuspended in cRPMI medium. The cells were then decanted into new tubes to remove remaining aggregated connective tissues. The cells were then counted in 0.2% trypan blue (Corning no. 25-900-CI, Manassas, VA) and plated in 24 well cell culture plates (Corning no. 3524, Kennebunk, ME) for subsequent experiments.

Cell Staining and Flow Cytometry

The suspension cells were collected, washed with DPBS supplemented with 5% fetal bovine serum and 0.1% sodium azide (FACS buffer), and stained with fluorescent lectins or fluorescent antibodies on ice for 30–60 min. In order of appearance, stains used in this study include 0.1 μg/mL of Alexa Fluor 647-labeled cyanovirin-N (CVN),27 4 μg/mL of Brilliant Violet 510 antimouse CD3 Antibody (Biolegend no. 100234, San Diego, CA), 2.5 μg/mL of PE/Cyanine5 antimouse CD19 Antibody (Biolegend no. 115509), 4 μg/mL of PE/Cyanine7 antimouse CD20 Antibody (Biolegend no. 150419), 2.5 μg/mL of PE antimouse CD22 Antibody (Biolegend no. 126111), 4 μg/mL of Brilliant Violet 421 antimouse/human CD45R/B220 Antibody (Biolegend no. 103251), 4 μg/mL of APC antimouse IgM Antibody (Biolegend no. 406509), 4 μg/mL of PerCP/Cyanine5.5 antimouse CD79b (Igβ) Antibody (Biolegend no. 132809), 2.5 μg/mL of Brilliant Violet 421 antimouse CD19 Antibody (Biolegend no. 115549), 1 μg/mL of SNA-FITC (Vector Laboratories no. FL-1301–2, Newark, CA), 5 μg/mL of MAL II-Cy5 (GlycoMatrix no. 21511110-1, Dublin, OH), 1:50 of antihuman IgM Antibody (Biolegend no. 314507), 1:50 of PE antihuman CD79b (Igβ) Antibody (Biolegend no. 341404), 10 μg/mL of FITC antihuman CD19 Antibody (Biolegend no. 302256). Labeled cells were washed and resuspended in 200 μL of FACS buffer. Flow cytometry was performed on a BD FACSAria Fusion cytometer (San Jose, CA). The collected data was analyzed using FlowJo v10 (Ashland, OR).

Calcium Flux

After 24 h of treatment, suspension cells were collected, washed with DPBS, and stained with 5 μM Fluo-4 AM (Invitrogen no. F14201 Eugene, OR) and 0.4 μg/mL of APC/Cyanine7 antimouse/human CD45R/B220 Antibody (Biolegend no. 103223) in DPBS for 30 min at room temperature. Labeled samples were washed in DPBS and resuspended in 450 μL of Hanks’ balanced salt solution (Gibco no. 14025, Grand Island, NY). Prior to loading samples onto the flow cytometer, cells were warmed to 37 °C for ∼2.5 min before baseline Fluo-4 measurement was taken for 30 s. Then, 10 μg of F(ab’)2 fragment antimouse IgM (Jackson ImmunoResearch Inc. no. 115-006-075, West Grove, PA) or antihuman IgM (Jackson ImmunoResearch Inc. no. 109-006-129) was added, and a stimulated measurement was collected for another 3 min. The collected data was analyzed using FlowJo on the kinetics platform.

Statistical Analysis

Statistical evaluation was performed using one-way or two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test using GraphPad Prism 7 (San Diego, CA).

Acknowledgments

We thank the National Institute of General Medical Sciences [P20 GM113117, P30 GM110761 and T32 GM008545 (to P.R.)] for supporting this work. We thank Dr. E. Go and the Synthetic Chemical Biology Core Facility at the University of Kansas for providing the MALDI-TOF service. This facility is supported by NIGMS grants P20GM113117 and P20GM103638. A.H. was supported by the Gretta Jean & Gerry D. Goetsch Scholarship at the University of Kansas. The graphical abstract was created using BioRender (BioRender.com).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00153.

  • Kifunensine treatment increased the surface expression of high mannose glycoproteins in B cells; effects of kifunensine and derivatives on Raji B cells; reaction scheme for the synthesis of kifunensine, JDW-II-004, and JDW-II-010 (PDF)

Author Contributions

A.H., J.D.G, C.J.B., and M.P.F. designed the study. A.H. conducted and analyzed the in vitro assays. S.E.K., and P.R. planned and performed the synthesis of the molecules. A.H. and M.P.F. wrote the original draft; and S.E.K., P.R., K.D.A., J.D.G., and C.J.B participated in the reviewing and editing. All authors have read and approved the final manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt2c00153_si_001.pdf (363.1KB, pdf)

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