Introduction

Carbohydrates are not only one of the three major components in the diet, along with protein and fat, but also constitute one of the three essential biological macromolecules in mammalian systems, that include nucleic acids and proteins. Besides playing an important role in providing energy and serving as building blocks for bigger macromolecules, carbohydrates are essential components for adding functions to proteins and lipids by forming glycoproteins and glycolipids, respectively. Glycoproteins and glycolipids are mostly embedded in cell surface membranes with unique fingerprint-like cell-specific signatures for distinct cells1. The carbohydrate moieties (glycans) can be used to interact with other cells via specific receptors (lectins)1.

Lectins are proteins with affinity to glycans and are produced in almost all life forms, including bacteria, plants, animals, and humans2. Mammalian lectins and glycans are known to be involved in a variety of biological processes such as cell-cell interactions3, signaling4, development5,6, immune responses7,8 and cancer9. Lectins produced by various pathogenic bacteria have been well studied and are known to play a crucial role in establishing a niche and evading the host’s defense systems10. As such, bacteria-binding lectins have been utilized as a tool to mediate the first-line defense against invading microorganisms11. However, lectins produced by non-pathogenic bacteria are poorly understood12.

Over 40 years ago, Fujita-Yamaguchi et al. identified a lectin produced by a non-pathogenic bacterium, Streptomyces sp. 27S513,14,15,16; this lectin specifically binds to blood-type B glycan(s) on human erythrocytes and causes hemagglutination. Therefore, this bacterial lectin was named Streptomyces hemagglutinin (SHA). While the original strain of Streptomyces sp. 27S5 has been lost, we successfully utilized the amino acid sequence of SHA to recombinantly reconstruct SHA protein (13.3 kDa)17. This recombinant SHA has been verified to bind to both human type B erythrocytes and to microbial Lactobacillus casei cells17. The cell wall of Lactobacillus casei is enriched for L-rhamnose18, which is a known glycan that SHA binds17. However, it remains unknown whether any other mammalian cell type, beyond human type B erythrocytes, displays SHA-binding glycans (SHA-BGs).

In humans, aberrant glycosylation is known to occur in essentially all types of cancers19,20,21, including pancreatic adenocarcinoma (PDAC)22,23. PDAC is one of the most aggressive malignancies with the worst prognosis among all cancers24. PDAC can arise from pancreatic acinar or ductal cells25, which are the two exocrine cell types that function by secreting digestive enzymes or transporting the enzymes into the duodenum, respectively. The glycan sialyl Lewis A (sLeA; CA19-9) has been shown as necessary for PDAC initiation26, is increased during tumor progression and has served as a clinical biomarker27. Although a connection of aberrant glycosylation with PDAC has been established9, a clear picture on glycans present in normal pancreatic exocrine cells is still far from complete28, which may have implication in future cancer diagnostics and treatment.

In this study, we began our efforts by testing whether SHA-BGs are present on normal pancreatic cells. Here, we find that SHA specifically targets pancreatic acinar cells, but not ductal or endocrine cells. This binding occurs in normal adult mice as well as during late stage embryogenesis (immediately prior to birth), but not in early pancreas development. SHA also specifically binds to acinar cells of the normal adult human donors with the B blood type. Our results indicate that SHA-BGs exist on normal human and murine pancreatic acinar cells and are developmentally regulated in mice. The mCherry-SHA that we produced has implications to serve as a biomarker and a useful tool to interrogate the glycan structures displayed on normal and pathological pancreatic acinar cells.

Materials and methods

Construction, expression, and purification of mCherry-SHA protein

The synthetic SHA gene was constructed as previously reported17. In short, the SHA homologous domain of the polysaccharide deacetylase (PDSL) gene from S. lavendulae was mutated at A108E to obtain the gene that encodes the amino acid sequence matching the original SHA protein isolated from Streptomyces sp. 27S5.

The final protein construct of mCherry-SHA is as follows: amino acid (AA) 1-236, mCherry29; AA 237–245, acidic linker; AA 246–378, SHA; and AA 379–384, poly-histidine (His6) tag. Acidic linker peptides are Gly-Asp-Glu-Val-Asp-Glu-Asp-Glu-Gly. DNA plasmids containing sequences encoding for mCherry, acidic linker, SHA followed by His6 tag in the vector pMK-RQ-Bs were generated by GeneArt services (ThermoFisher Scientific, Waltham, MA). Subsequently, restriction enzymes NcoI-HF/XhoI (New England Biolabs, Ipswich, MA) were used to cut the fragment containing the mCherry-acidic linker-SHA and then cloned into the pET28 expression vector equipped with a His6 tag (Cat. No. 69864-3; Novagen, Madison, WI), a plasmid which we named “pET28-mCherry-SHA”. Sequence fidelity was verified by Sanger sequencing. The pET28-mCherry-SHA was transfected into BL21 (DE3) E. coli competent cells and the fusion protein expressed after the induction by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25 ℃ overnight.

The culture was centrifuged at 3500×g for 20 min to obtain cell pellet, which was collected, washed in ice-cold PBS, and centrifuged again at 3500×g for 20 min. For pellet from 1 L of culture, 50 mL of B-PER bacterial protein extraction reagent (ThermoFisher Scientific) supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher Scientific), 6 M urea, and 1 mM phenylmethylsulfonyl fluoride (PMSF) was added to produce a lysate. The lysate was spun at 17,000×g for 30 min and supernatant was filtered through a 1 μm and then a 0.22 μm filter.

The mCherry-SHA protein was purified using a His tag-specific nickel (Ni)-NTA column (ThermoFisher Scientific) as follows. The lysate was incubated with Ni-NTA beads in 20 mM Tris-HCl buffer, pH 8.0, containing 300 mM NaCl, 40 mM Imidazole, and 10 mM β-mercaptoethanol in a cold room with continuous mixing for 15 min, passed through a gravity flow column, and washed with decreasing concentrations of urea (5, 3, 1 M urea) in the presence of 1 M galactose and 10 mM β-mercaptoethanol to permit refolding. Finally, renatured mCherry-SHA was eluted with 400 mM imidazole from the Ni-NTA column. Activity of the recombinant protein was verified by binding to and eluting from a Gum Arabic affinity resin as described previously (12). mCherry-SHA was concentrated using a Centricon YM10 centrifugal filter (EMD Millipore, Burlington, MA) and purified by fast protein liquid chromatography (FPLC) in 20 mM Tris-HCl buffer, pH 8.0, containing 300 mM NaCl, 40 mM Imidazole, and 10 mM β-mercaptoethanol on a Superdex 75G gel filtration column (GE Healthcare, Chicago, IL).

To generate the control mCherry protein, subcloning of the pET28-mCherry-SHA was performed to isolate the mCherry DNA fragment, and clone back to the pET28 vector with a His6 tag. Subsequently, the mCherry/His6 protein was expressed in BL21(DE3) E. coli and purified by Ni-NTA affinity chromatography in 20 mM Tris-HCl buffer, pH 8.0, containing 300 mM NaCl and 40 mM Imidazole as described above, but under non-denaturing conditions without the presence of urea and 1 M galactose.

To confirm protein sequences of mCherry-SHA and the control mCherry, the purified and concentrated forms of both proteins were separated on a NuPage 4–12% Bis-Tris protein gel (ThermoFisher Scientific) followed by an in-gel digestion for mass spectrometric analysis as previously described17.

Mice

All animal experiments were approved by the Institutional Animal Care and Use Committee at the City of Hope (protocol #11017).; experiments were performed in accordance with the relevant guidelines and regulations, and reporting consistent with the ARRIVE guidelines30. C57BL/6J (B6) mice (both sexes; The Jackson Laboratory, Bar Harbor, ME; RRID: IMSR_JAX:000664) were used in this study. The age of mice used in each experiment is stated in the figure legend. Mice were group-housed by sex with up to five siblings per cage and with ad libitum food and water. Cages were held at 22 °C, changed once every 2 weeks, and regularly monitored for virus and parasite infection. Euthanasia was performed by CO2 inhalation without any other chemical agent, and with cervical dislocation as a secondary method following asphyxiation. For timed pregnancies, presence of a vaginal plug in the morning was defined as embryonic day (E) 0.5. Pregnant mice were euthanized before embryos were collected for study.

Human pancreas tissue slides

Human pancreas tissues from adult cadaveric donors were obtained by the Southern California Islet Cell Resource (SC-ICR) Center at the City of Hope. In short, donated cadaveric pancreases were shipped to SC-ICR for the purpose of isolation of islets. Prior to islet isolation, a portion of the pancreatic tissue was excised, fixed in 10% formalin, paraffin embedded, and archived. Tissue blocks were then sectioned at a thickness of 5 μm. All tissues used in this study had been consented for research by the close relatives of the donors. For the studies described in this work, SC-ICR provided formalin-fixed, paraffin-embedded pancreas tissue slides to H.T.K., along with deidentified donor information. This process was reviewed and approved by the Institutional Review Board at the City of Hope as Not Human Subjects Research (Protocol # 23728). All methods were performed in accordance with the relevant guidelines and regulations.

Immunofluorescence staining

L. casei immunofluorescence staining was performed following a previously established protocol17. Briefly, L. casei cells were inoculated into 400 ml of DifcoTM Lactobacilli MRS broth (ThermoFisher Scientific) at a concentration of 106 cells/ml. Cells were grown for 16 h at 37 °C, harvested by centrifugation, and washed three times with 1× phosphate-buffered saline (PBS). Cells were resuspended in 5 ml of 70% ethanol and incubated at 22 °C for 30 min under continuous rotation. After ethanol treatment, cells were washed three times with 1× PBS. To reduce nonspecific binding, cells were incubated with 5 ml blocking buffer containing 3% BSA in PBS and Nonidet P-40 (0.5%) for 30 min. This was followed by 1 h of incubation with 5 µg/mL of mCherry–SHA or mCherry diluted in 1× PBS at pH 7.4. After washing three times with 1× PBS, cells were resuspended in 1 ml of 1× PBS containing 10% glycerol. Cells were counterstained with DAPI (50 nM) and examined using a Zeiss Observer II system. Fluorescence images were prepared using Image-Pro Plus (RRID: SCR_007369) and ZEISS ZEN software (RRID: SCR_013672).

For frozen section of murine embryonic pancreas, dissected pancreases were fixed in 4% paraformaldehyde at 4 °C overnight, washed with 1× PBS (pH = 7.4–7.6), cryoprotected at 4 °C overnight in 30% sucrose dissolved in 1× PBS, followed by frozen embedding using Optimal Cutting Temperature (OCT) compound (ThermoFisher Scientific; 23-730-571). Frozen blocks were sectioned (10 μm thickness) onto glass slides (Fisher Scientific) and stored at −80 °C. Subsequently, slides were thawed at room temperature and then immersed in 1× PBS before the staining procedure.

To obtain formalin-fixed, paraffin-embedded (FFPE) tissue sections, pancreases from mice aged E18.5- to 35-week-old were dissected, fixed in 10% formalin for 24 to 72 h, paraffinized on a Tissue-Tek VIP Vacuum Infiltration Processor (SAKURA, Torrance, CA), sectioned at 5 μm thickness onto glass slides and stored.

To stain FFPE samples from murine or human pancreas tissues, slides were prepared by baking at 56 °C for 3 h, followed by de-waxing in 3 xylene baths and rehydrating in ethanol, as described previously31. Antigens were retrieved using IHC-TekTM Epitope Retrieval Steamer Set (IHC WORLD, Ellicott City, MD) for 45 min at 80 ℃ in sodium citrate buffer (pH = 5.5). From this point, slides from both FFPE and frozen samples underwent the same procedure.

Slides were washed with PBSX (0.1% Triton X-100 in 1× PBS), permeabilized in 0.3% Triton X-100 in PBS for 30 min and blocked with a PBS-based buffer containing 10% donkey serum and 1× Universal Power Block (BioGenex, Fremont, CA) in 0.1% Triton X-100 for 2 h at room temperature (RT). Slides were incubated with primary antibodies and either mCherry-SHA (700 µM)or mCherry (700 µM) diluted in blocking buffer at 4 °C overnight. Next, slides were washed, treated with appropriate secondary antibodies at RT for 2 h, washed and treated with Vector® TrueVIEW™ Autofluorescence Quenching Kit (VectorLabs, Newark, CA). Nuclei were visualized by incubating for 15 min with 0.1 µg/ml DAPI in PBSX. Finally, slides were washed with PBSX and treated with VECTASHIELD Antifade Mounting Medium (VectorLabs) with a coverslip placed on top of the tissue and sealed with nail polish. Slides were observed using a ZEISS LSM 880 confocal microscope, ZEISS Observer II, or ZEISS Axioscan 7 (Carl-Zeiss, Oberkochen, Germany) at City of Hope’s Light Microscopy Digital Imaging Core facility. Images were prepared using ZEISS ZEN 3.6 lite software (Carl-Zeiss), and figures were created in Photoshop (RRID: SCR_014199; Adobe, San Jose, CA). The antibodies used in this study are listed in Table 1.

Table 1 List of antibodies.
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SHA intensity quantification

Scanned images of pancreas sections shown in Figs. 3 and 5 were analyzed for fluorescence intensity using QuPath-0.3.232. Individual cells across the entire pancreas section were first detected using DAPI+ nuclei. To identify all Cpa1+ cells, a setting threshold based on the average Cpa1+ cytoplasm intensity was applied. From each of the identified Cpa1+ cells on one pancreas, the intensity of the mCherry-SHA signal or the mCherry signal from the adjacent slide was determined and averaged. Subsequently, the ratio between the mean intensity of mCHerry-SHA compared to mCherry from Cpa1+ cells from each pancreas was calculated, Log2 transformed and presented.

Immunohistochemistry staining

Colorimetric immunohistochemistry staining was conducted on FFPE slides from murine or human pancreas tissues. Slides were prepared as described above for baking, rehydration, and antigen retrieval. Slides were washed with PBSX, permeabilized as above, incubated with 1% H2O2 for 10 min, and then incubated with blocking buffer for 1 hr at RT. Slides were washed with PBSX and incubated with mCherry-SHA (1400 µM) or mCherry (1400 µM) diluted in blocking buffer at 4 °C overnight. Next, slides were washed with PBSX and incubated with biotin-labeled anti-6×-His Tag monoclonal antibody (clone HIS.H8; Thermo MA1-21315-BTIN) in blocking buffer at 4 °C overnight. Slides were then washed with PBSX, treated with horseradish peroxidase (HRP)-conjugated streptavidin (Vector Labs, #SA-5004-1) for 1 h, washed and treated with 3,3′-diaminobenzidine (DAB) solution (Vector Labs, #SK-4100) for 30 min at room temperature. Slides were counter-stained with Hematoxylin (Abcam, #ab245880) for 10 min and treated with Bluing Reagent for 15 s. Slides were then mounted with VectaMount (Vector Labs, H-5700) with a coverslip placed on top of the tissue. Slides were observed using a ZEISS Observer II at City of Hope’s Light Microscopy Digital Imaging Core facility. Images were prepared using ZEISS ZEN 3.6 lite, and figures were prepared in Photoshop.

Statistical analysis

One-way ANOVA was used to determine significance. * indicates p < 0.05, and ** p < 0.005.

Results

Production and characterization of recombinant SHA protein tagged with mCherry

The original Streptomyces sp. strain 27S5 has been lost and therefore no genomic information is available. In our previous study, we utilized mass spectrometry to decipher the amino acid sequence of SHA, which was purified and kept frozen at −80 ℃ for over 40 years17. Using the genomic databases, we identified a matching hypothetical protein in the genome of Streptomyces lavendulae ATCC 1415817. This hypothetical SHA homologue has over 99% amino acid sequence homology to that of the authentic SHA, with just one amino acid difference: residue 108 is a glutamic acid in authentic SHA whereas an alanine in the Streptomyces lavendulae homologue17.

In order to visualize SHA-BGs present in mammalian cells, we initially constructed and produced a green fluorescence protein (GFP)-SHA fusion protein. The resulting GFP-SHA protein has been characterized and is confirmed to be capable of binding to known targets, Lactobacillus casei (strain Shirota) and human blood type B erythrocytes17. However, GFP-SHA was highly hydrophobic, resulting in aggregated forms, thus precluding effective imaging analysis.

We therefore designed and produced a mCherry-SHA fusion protein in this study (Fig. 1A). The control protein mCherry is depicted in Fig. 1B. Next, Lactobacillus casei was fixed on glass slides and stained with mCherry-SHA or mCherry control. As expected, mCherry-SHA (Fig. 1C) but not the control mCherry (Fig. 1D) was found to positively stain Lactobacillus casei, as analyzed by fluorescence microscopy.

Fig. 1
figure 1

Construction and validation of the mCherry-SHA protein for use in immunofluorescence microscopy analysis. (A,B) Schematic depiction of the mCherry-SHA fusion protein (A) and the control mCherry protein (B). (C) The positive control cells, Lactobacillus casei, were fixed and stained with mCherry-SHA and DAPI (nuclei). (D) Lactobacillus casei were stained with the control mCherry and DAPI. Bars = 10 μm. Images were obtained from a ZEISS Observer II microscope.

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mCherry-SHA specifically targets acinar cells but not ductal or endocrine cells in the adult murine pancreas

Next, we tested whether SHA could bind to pancreatic cells of normal adult mice. FFPE pancreas tissue slides from 15 weeks old mice were co-stained with mCherry-SHA and antibodies against pancreatic lineage markers for acinar (Cpa1)33 and ductal (Krt19) cells34. Fluorescence microscopy analysis revealed that mCherry-SHA signal (red) overlapped with Cpa1 (white); however, islet area (outlined by a solid white line) and Krt19-positive cells (green) were negative for mCherry-SHA signal (Fig. 2A). The control mCherry staining on a sequential slide showed minimal signal in the adult murine pancreas (Fig. 2B). To further confirm staining specificity, primary antibodies against Cpa1 and Krt19, as well as mCherry-SHA/mCherry, were omitted, which showed the background in the respective fluorescence channels (Fig. S1A).

Fig. 2
figure 2

Pancreatic acinar, but not ductal or islet cells, exhibit positive staining with mCherry-SHA in mice. (A) Co-staining of adult (15 weeks-old) murine pancreas with mCherry-SHA, Cpa1 (an acinar cell marker) and Krt19 (a ductal cell marker). Representative photomicrographs from formalin-fixed paraffin-embedded pancreas sections are shown. Bars = 50 μm. (B) Control mCherry was used instead of mCherry-SHA. Bars = 50 μm. (C) Magnified images for triple-staining with mCherry-SHA, Cpa1 and Krt19. Two insets are further magnified in the right panels, revealing the localization of mCherry-SHA on the acinar cell membrane and in the cytoplasm. The arrow indicates a ductal lumen positively stained with Cpa1, an acinar cell-secreted enzyme transported by the ducts. Bars = 10 μm. Islet areas are indicated by solid white lines in (A) and (B). Images were obtained from a ZEISS LSM 880 confocal microscope.

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To examine the staining pattern in greater detail, higher magnification of the photomicrographs in Fig. 2A confirmed overlapping regions of Cpa1 and mCherry-SHA, which lacked Krt19 (Fig. 2C). Furthermore, mCherry-SHA was localized to the cell membrane of acinar cells, but signals in cytoplasm were also observed (Fig. 2C, inset 1). A bi-nucleated Cpa1+ cells, which is a known feature for some pancreatic acinar cells35, also displayed mCherry-SHA binding signal (Fig. 2C, inset 2). Together, these results demonstrate that SHA specifically interacts with acinar cells on cell membrane and cytoplasm in the adult murine pancreas.

Staining of mCherry-SHA to pancreatic acinar cells is inhibited in the presence of L-rhamnose

The identity of the SHA-BGs on mammalian acinar cells is currently unknown. Therefore, to further determine the binding specificity of mCherry-SHA to acinar cells, we performed a competition study using the bacteria-specific monosaccharide, L-Rhamnose, on sequential pancreas slides (Fig. 3A–D). Consistent with the finding from Fig. 2, the mCherry-SHA signal co-localized with Cpa1 (Fig. 3A). The addition of 25, 100 and 400 mM L-Rhamnose reduced mCherry-SHA signal (Fig. 3B–D). The mCherry control again showed the background staining (Fig. 3E). The intensity of the red fluorescence signal within Cpa1+ acinar cells was quantified, and the ratio between paired mCherry-SHA staining and mCherry from the sequential section was determined; a significant decrease of mCherry-SHA signal was observed with the addition of all concentrations of L-Rhamnose tested (Fig. 3F). These results confirmed the specificity of the recombinant SHA protein.

Fig. 3
figure 3

The mCherry-SHA binding signal on acinar cells is competitively blocked by the addition of L-Rhamnose. (A–D) Competition of mCherry-SHA signal in the acinar cells with increasing doses of L-Rhamnose (0 to 400 mM). The arrows indicate a ductal lumen positively stained with Cpa1 and mCherry-SHA, which is also reduced by L-Rhamnose competition. This suggests that SHA-BGs are secreted into ducts. (E) Control staining using mCherry and Cpa1. Islet regions are delineated by solid white lines. Bars = 50 μm. Images were obtained from a ZEISS LSM 880 confocal microscope. (F) Quantification of red fluorescence signal from mCherry-SHA in Cpa1-positive cells, normalized to mCherry signal in Cpa1-positive cells of the adjacent slide. Each dot represents a mouse pancreas. One-way ANOVA was used to determine significance. *P < 0.05. **P < 0.005.

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mCherry-SHA recognizes murine acinar cells at E18.5, but minimally at E12.5 and E15.5 during embryogenesis

Glycosylation of various proteins and lipids are known to occur during development in specific spatial and temporal patterns5,6. We therefore examined the kinetics of SHA-BGs during pancreas development. Staining experiments were performed on frozen sections obtained from embryonic day (E) 12.5, 15.5, and 18.5 murine pancreas. An anti-Sox9 antibody that can recognize both ductal and multipotent progenitor cells during pancreas development36 was used to aid the identification of the early pancreatic buds. Triple-staining of mCherry-SHA, Cpa1, and Sox9 revealed that mCherry-SHA binding was minimal in E12.5 and E15.5 pancreas (Fig. 4A,B). In contrast, mCherry-SHA signal was clearly detected in the Cpa1+ acinar cells but not Sox9+ ductal cells of E18.5 pancreas (Fig. 4C). The control staining with mCherry on sequential slides showed minimum background in all ages examined (Fig. S1B). These results suggest that expression of SHA-BGs on acinar cells is developmentally regulated.

Fig. 4
figure 4

The mCherry-SHA binding signal is minimal between E12.5 and E15.5 but becomes detectable in acinar cells at E18.5 during murine pancreas development. Co-staining of murine pancreas with mCherry-SHA, Cpa1 and Sox9 from mice aged (A) embryonic day (E) 12.5, (B) E15.5, and (C) E18.5. Representative photomicrographs from paraformaldehyde-fixed frozen pancreas sections are shown. Bars = 50 μm. Images were obtained from a ZEISS Observer microscope.

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mCherry-SHA recognizes murine acinar cells aged between E18.5 to 35 weeks old

To extend the temporal analysis, we examined FFPE sections from mice at multiple stages of pancreas development: E18.5, postnatal (P) day 8–10, young adult week (W) 13–15, and older adult W25-35. Again, mCherry-SHA specifically stained Cpa1+ acinar cells, excluding ductal or endocrine cells, across all ages examined (Fig. 5A–D). The mCherry control exhibited minimal background staining in the acinar cells once again (Fig. S2A–D). We determined whether the signal intensity of mCherry-SHA changes as the mice age and found no significant difference between the ages (Fig. 5E). Interestingly, at E18.5, large variation was observed in the mCherry-SHA intensity among the 8 mice examined (Fig. 5E; first bar), suggesting a transitional time for SHA-BG expression during development.

Fig. 5
figure 5

Pancreatic acinar cells are recognized by mCherry-SHA in mice aged between E18.5 to 35 weeks old. Co-staining of murine pancreas with mCherry, Cpa1 and Krt19 on (A) E18.5, (B) postnatal day (P) 10, (C) 15 weeks and (D) 25 weeks old mice. Representative photomicrographs from formalin-fixed paraffin-embedded pancreas sections are shown. (E) Quantification of red fluorescence signal from mCherry-SHA in individual Cpa1-positive cells, normalized to the mCherry signal detected in individual Cpa1-positive cells on a sequential section. Each dot represents a mouse pancreas. E18.5, n = 8; p8–p10, n = 10; 13–15 weeks, n = 3, and 25–35 weeks, n = 3. One-way ANOVA was used to determine significance. ns: not significant. (F) Representative brightfield images of mCherry-SHA (left panel) or mCherry staining (right panel), identified by anti-6×-His-tag antibody and visualized by DAB (brown) and hematoxylin staining (blue). Bars = 100 μm. Islet regions are indicated by solid white (A–C) or black (E) lines. Images were obtained from a ZEISS LSM 880 confocal microscope (A–D) or Zeiss Observer II (F).

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To further confirm the specificity of mCherry-SHA binding to the acinar cells, we performed immunohistochemistry staining using antibody against the 6×-His tag, which had been engineered into both the mCherry-SHA fusion protein (Fig. 1A) and the mCherry control (Fig. 1B). Subsequently, DAB (brown color) was used as a substrate to visualize the 6×-His staining. Hematoxylin was used as a counter stain. Again, 6×-His tagged with the mCherry-SHA, but not that with mCherry, was found to bind to the pancreatic acinar cell area but not the islets (Fig. 5F). Taken together, these results demonstrate that SHA-BGs are formed as early as E18.5 and are maintained for at least eight months after birth in mice.

mCherry-SHA specifically targets human adult pancreatic acinar cells from donors with blood B but not A or O types

To extend our findings from mice to humans, we examined the SHA binding pattern on FFPE pancreas tissue slides obtained from cadaveric donors without apparent diseases. Donor characteristics are summarized in Table 2. In type B donor tissue, the mCherry-SHA signal once again overlapped with the CPA1+ acinar cells, while islet cells did not display a positive signal (Fig. 6A, upper panels). Magnified photomicrographs further revealed co-localization of SHA signal with CPA1 but not KRT19 (Fig. 6A, lower panels). In contrast, mCherry-SHA did not stain CPA1+ acinar cells in donors with blood type A (Fig. 6B) or type O (Fig. 6C). Control mCherry showed background staining (Fig. S3). Out of the nine donors tested, only acinar cells from blood type B donors (N = 3) exhibited positive staining with mCherry-SHA, whereas those from type A (N = 2) or O donors (N = 4) did not (Fig. 6D; Table 2). DAB and hematoxylin staining using anti-6×-His antibody further confirmed that the mCherry-SHA fusion protein, but not mCherry, binds to the pancreatic acinar cell area (Fig. 6E). Taken together, these results demonstrate that SHA also targets human acinar but not islet or ductal cells; however, only blood type B donor acinar cells can be stained positively with SHA.

Table 2 Human donor characteristics and results on SHA staining in acinar cells.
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Fig. 6
figure 6

The mCherry-SHA targets human pancreatic acinar cells from donors with blood type B, but not from those with type A or O. Archived formalin-fixed paraffin-embedded human pancreas tissues were examined. Slides were triple stained with mCherry-SHA, CPA1, and KRT19. Representative photomicrographs from a donor of blood type B (A), type A (B), and type O (C) are shown. Insets in (A) show magnified images with various combinations of staining. (D) Summary of the 9 different human donor tissues examined in this study. pos. positive, neg. negative. (E) Representative brightfield images of mCherry-SHA (top panel) or mCherry staining (bottom panel), identified by anti-6×-His antibody and visualized by DAB (brown) and hematoxylin staining (blue). Bars = 100 μm. Islet regions are indicated by solid white (A–C) or black (E) lines. Images were obtained from a ZEISS LSM 880 confocal microscope (A–C) or Zeiss Observer II (E). Additional human donor characteristics are found in Table 2.

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Discussion

In this study, we demonstrate that a lectin (named SHA) produced and secreted by a non-pathogenic Streptomyces sp. exhibits the capacity to bind to acinar cells specifically, excluding ductal or endocrine cells, in the normal pancreas of mice and humans with blood type B. To date, only a limited number of bacterial lectins have been identified12,14,37,38, many of which are derived from opportunistic or pathogenic bacteria, such as S. pneumoniae from the Streptococcus genus. In contrast, SHA is derived from a species belonging to the Streptomyces genus. Bacteria in this genus are primarily found in soil and decaying vegetation, serving as a valuable source of antibiotics and are infrequently pathogenic to humans.

This study finds SHA as the first bacterial lectin that recognizes pancreatic acinar cells. Prior studies have identified two plant lectins that also specifically recognize acinar but not ductal or endocrine cells in the pancreas. Ulex europaeus agglutinin I (UEA I) can bind to human39 and rat40 pancreatic acinar cells. However, it should be noted that UEA I has a strong affinity to human endothelial cells as well41. Peanut agglutinin (PNA) shows affinity for murine pancreatic acinar cells42, as well as for rat glomus cells in the carotid bodies43 and a subpopulation of murine lymphocytes44. Whether SHA recognizes other organ cell types requires further investigation.

The gut microbiome has been linked to several chronic diseases, including pancreatic diseases45. Recently, Cohen et al. sequenced microbes isolated from human stool samples and identified two lectins, Cbeg4 and Cbg5 secreted from the Bacteroides sp., that can bind and activate human myeloid cells in vitro and secrete pro-inflammatory cytokines such as IL-1b and IL-612. Therefore, the gut bacteria-secreted lectins can provide a functional link to human diseases via immune cells. Cohen et al. also find 5 proteins made by gut Streptomyces sp.: they are sialidase domain-containing protein, xanthan lyase, FAD dependent oxidoreductase, Rhs family protein, and glycoside hydrolase family 512. However, these proteins are not lectins. Regardless, based on their microbiome sequencing data, Cohen et al. anticipate the existence of thousands of additional novel bacterial lectins, awaiting further characterization. Thus, our current discovery represents a small step forward in identifying a bacterial lectin that can bind to primary murine and human acinar cells. However, the biological consequence of the binding of SHA to acinar cells requires further investigation.

We observe that only pancreatic acinar cells from blood type B donors, and not from O or A types, exhibit positive staining with mCherry-SHA (Fig. 6). This is not surprising because it has been shown that human pancreatic acinar cells express the same respective ABO epitopes46. For example, a mouse monoclonal antibody (clone 89-F) specific for the human B blood type carbohydrate moiety can also recognize pancreatic acinar but not endocrine or ductal cells46. This raises the possibility that the B antigen on erythrocytes, recognized by the 89-F antibody, may be identical to the SHA-BG on human acinar cells, which requires further investigation. In our previous study using a glycan array analysis17, SHA has been found to bind to known blood B type-specific oligosaccharides, such as trisaccharide Gal-α-1,3-(Fuc-α-1,2)-Gal-β- and tetrasaccharide Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-. In addition, SHA can bind to closely-related glycans such as Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-; Gal-α-1,4-Gal-β-1,4-Glc-β-; and Gal-α-1,4-Gal-β-1,4-GlcNAc-β-. Thus, SHA may also recognize additional glycan or glycans on acinar cells other than the blood group B-specific 89-F epitope.

Consistent with the expectation that glycosylation occurs during development in specific spatial and temporal patterns5,6, we find that SHA-BGs start to be expressed on acinar cells around E18.5 and thereafter (Fig. 5), but not E15.5 or E12.5 (Fig. 4). Acinar cell maturation can be indicated by zymogen granule formation47. Consistent to developmental regulation of glycans, the glycosylation of a zymogen granule membrane protein gp300 is also age dependent48.

PDAC is one of the most aggressive malignancies with the worst prognosis among all cancers, partly because patients are usually diagnosed at later stages49. PDAC can be caused by improper glycosylation, which plays a role in disease initiation and progression9,50,51. For example, genes such as Cosmc (core 1 β3GalT specific molecular chaperone) and C1GALT1 (core 1 β1-3 galactosyltransferase) are required to synthesize the Core 1, T antigen (Galβ1-3GalNAc-α1-O-Ser/Thr), which is a basis to build complex O-glycans required for proper cellular functions52. Truncated O-glycans resulting from hypermethylation of the Cosmc gene in PDAC cells enhance tumor aggressiveness by inducing epithelial-mesenchymal transition (EMT) and stemness properties51. Our prior glycan array results demonstrated that Galβ1-3GalNAc-α-configuration has no affinity to SHA17, suggesting that Core 1, T antigen is not a receptor for SHA. In addition to the truncated O-glycans, an involvement of sialyl modifications in glycans during PDAC progression has also been suggested50.

Currently, a prevalent clinical biomarker used in the management of pancreatic cancer is the serum marker CA19-953, which also plays a role in PDAC tumor progression26. While the CA19-9 assay is used routinely for monitoring treatment response54, concerns have been raised about its sensitivity and specificity as a diagnostic biomarker53. Thus, there is an urgent need to identify additional biomarkers to enable diagnosis and treatment of PDAC. Whether SHA-BGs may serve as a new biomarker for PDAC is currently unknown and is under active investigation.

In summary, we have demonstrated that a lectin produced by Streptomyces sp. 27S5 is capable of binding to acinar cells but not ductal or endocrine cells in the normal pancreas of mice and humans with blood type B. The binding of SHA to glycans is developmentally regulated, as evidenced by minimal binding in early murine pancreas, followed by positive binding in acinar cells from E18.5 and onwards in mice. Our discovery of SHA’s interaction with normal murine and human pancreatic acinar cells holds potential applications in basic research, serving as both a biomarker and a novel tool to aid in defining and studying the effects of specific glycans on mammalian cells that SHA binds to.