Aberrant environment and PS-binding to calnuc C-terminal tail drives exosomal packaging and its metastatic ability.

The characteristic features of cancer cells are aberrant (acidic) intracellular pH and elevated levels of phosphatidylserine. The primary focus of cancer research is concentrated on the discovery of biomarkers directed towards early diagnosis and therapy. It has been observed that azoxymethane-treated mice demonstrate an increased expression of calnuc (a multi-domain, Ca2+- and DNA-binding protein) in their colon, suggesting it to be a good biomarker of carcinogenesis. We show that culture supernatants from tumor cells have significantly higher amounts of secreted calnuc compared to non-tumor cells, selectively packaged into exosomes. Exosomal calnuc is causal for epithelial-mesenchymal transition and atypical migration in non-tumor cells, which are key events in tumorigenesis and metastasis. In vitro studies reveal a significant affinity for calnuc towards phosphatidylserine, specifically to its C-terminal region, leading to the formation of "molten globule" conformation. Similar structural changes are observed at acidic pH (pH 4), which demonstrates the role of the acidic microenvironment in causing the molten globule conformation and membrane interaction. On a precise note, we propose that the molten globule structure of calnuc caused by aberrant conditions in cancer cells to be the causative mechanism underlying its exosome-mediated secretion, thereby driving metastasis.


INTRODUCTION
Cancer is a complex disease that leads to the formation of tumors through uncontrolled growth and the spread of abnormal cells [1].The future of cancer pathology is solely dependent on the identification of biomarkers for the accurate detection and management of disease at an early stage.Intense molecular analysis of these tumors has led to the identification of the atypical expression of certain proteins.Identification and characterization of these molecules present us with potential cancer biomarkers [2].Advances in proteomics have propelled the discoveries of numerous potential biomarkers for diverse cancers.However, a precise knowledge of the physiological basis and mechanism of biomarker development is often missing.
Understanding the causal biochemical and molecular biological factors that underlie abnormal expression levels of these biomarkers is of utmost importance for early detection and therapeutic intervention [3].One group of such abnormally expressed proteins belongs to the Ca 2+ -binding protein family [4].Expression levels of calnuc or nucleobindin1 (Nucb1), a multi-domain Ca 2+binding protein, have been observed to be elevated in tumors of the stomach, lymphocytes, and colon, while its secretion has been reported in AtT20 pituitary tumor cells [5][6][7][8][9].In addition to the involvement of calnuc in the pathophysiology of cancer, autoantibodies against calnuc have been detected in the sera of colon carcinoma patients [6].However, the relationship between its expression levels and increased secretion in tumors is unclear.Our results provide an understanding of the role of calnuc in cancer prognosis.
The importance of calnuc in tumorigenesis also emanates from the fact that it is expressed ubiquitously and implicated in several physiological activities, including cell signaling, stress response, receptor sorting, inflammation, and apoptosis [10,11].Besides Golgi localization, calnuc is also targeted to other cellular organelles [10].The varying pH among these organelles [12] highlights calnuc's ability to withstand a wide pH range for its diverse functions.
An important notable fact is that cancer cells have an unusual acidic environment and high levels of phosphatidylserine (PS) [13,14].
Increased secretion of calnuc is described in certain tumors (pancreatic and head & neck tumors, colon carcinoma [6], lymphoma [8], gastric adenocarcinoma [7], pituitary [9,10]), and here, we present a novel mechanism of calnuc's action in tumorigenesis.We demonstrate that the aberrant microenvironment in cancer cells promotes calnuc interaction with membranes, 3 consequently promoting its tumor-specific exosomal packaging (PS-rich membrane-bound extracellular vesicles) [15] and release to the extracellular space.Unlike exosomes of non-tumor origin (involved in normal physiology), tumor-derived exosomes embody rich biological cargo that stimulates proliferation, metastasis, immune resistance, and angiogenesis [16].Accordingly, we establish that exosomal calnuc is responsible for epithelial-mesenchymal transition (EMT) and augments the migration of non-tumor cells, signifying its potential role in metastasis.Our study illustrates that the PS-mediated membrane association of calnuc through a molten globule state, which occurs as a result of a decrease in microenvironmental pH.A similar pH-dependent adoption of molten globule conformation is observed in calnuc from Drosophila melanogaster (DmCalnuc, 45% amino acid identity, and a similar domain organization), emphasizing its conserved physiological and functional significance.We demonstrate unequivocally that the PSinteraction-mediated exosomal packaging facilitates exosomal secretion of calnuc as a result of aberrant tumor cell microenvironment, and it acts as a metastasis inducer.This work, therefore, alludes to the possibility of new modes of cancer therapy by (i) targeting expression levels of calnuc and (ii) designing small molecules that use calnuc as a target.

Chemicals and Reagents
All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless mentioned.Buffers were prepared in decalcified Chelex 100 (Bio-Rad, Hercules, CA, USA) purified Milli-Q water.DOPC, DOPS, and DOPE lipid solutions in chloroform, extruder, and polycarbonate membranes were purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Immunostaining of calnuc on mice colon tissues
Formalin-fixed paraffin-embedded sections on coated slides from mice colon samples were gifted by Dr. Anita Sjölander (Lund University, Sweden), and were used to ascertain the expression of calnuc using immunohistochemistry.The mice experiments were carried out as per the protocol and published earlier [17].Briefly, 6-8-week-old female C57BL/6J (B6/J) mice were injected with 10 mg/kg azoxymethane (AOM) on day 0, followed by two 5-day cycles of 2% dextran sodium sulfate (DSS) in drinking water with two weeks recovery period between the cycles with an end-point of 90 days (CAC end-point).C57BL/6J (B6/J) mice treated with AOM/DSS were sacrificed on day 31 and 90 after AOM/DSS treatment.The colons from the Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016animals were removed, fixed with formalin and paraffin-embedded sections were prepared on coated slides.To analyze calnuc expression, slides were deparaffinized in graded alcohols, hydrated and incubated with mouse anti-calnuc antibody (1:100), and washed and developed using HRP-conjugated secondary antibody (Bond Polymer Refine Detection, Leica, Wetzlar, Germany).Slides were scored for calnuc expression by a veterinary pathologist & Q-scores were computed.Immunostaining was observed in Leica DM2000 LED upright brightfield microscope, 10X/0.25-NAobjective at room temperature, in Leica MC170 HD camera, and the image was acquired using Leica Application Suite.

Cell Culture and Antibodies
Fetal buccal mucosa (FBM) cell line is an immortalized and non-transformed cell line (a kind gift from Milind Vaidya, ACTREC, Tata Memorial Centre, Navi Mumbai, India).The human oral squamous cell carcinoma cell line (SCC131) was a kind gift from (Susanne M.

Collection of conditioned media
Cells of interest were grown to 70% confluence in corresponding media and supplements.
Cells were washed thoroughly with phosphate-buffered saline (PBS) before switching to serumfree RPMI media and grown for 24 h.The harvested conditioned media was collected and concentrated with Amicon centrifugal filter devices (Millipore Corporation, Bedford, MA, USA), and concentrated supernatants were analyzed using western blotting.

Exosome Isolation
Exosomes were isolated from the conditioned media (collected as mentioned above) using Total Exosome Isolation Reagent (Thermo Scientific, Waltham, MA, USA) as per the manufacturer's protocol.The size of isolated exosomes was identified using dynamic light scattering [18].The isolated exosomes were washed in 1X PBS and further purified by ultracentrifugation at 100000 xg for 90 min at 4 °C.Exosomes were resuspended and lysed in 1X RIPA buffer, and protein estimation was done by Bradford assay.The presence of calnuc in exosomal protein extracts was confirmed by western blotting.

Western Blotting
Cells grown in serum-free media were lysed in radioimmunoprecipitation (RIPA) buffer, and protein concentration was estimated by the Bradford method.75 μg of total lysate was loaded onto 10% SDS polyacrylamide gel and transferred to nitrocellulose membrane (Membrane Solution, Kent, WA, USA).The membrane was blocked with 5% skimmed milk in Tris-buffered saline with Tween-20 (1X TBST) for 1 h.Blots were incubated with corresponding primary antibody overnight.Blots were washed thoroughly with 1X TBST and incubated with 6 conjugated secondary antibody for 1 h at room temperature.Followed by a thorough wash with 1X TBST, blots were developed with commercial ECL reagent in ChemiDoc (Bio-Rad, Hercules, CA, USA).

Exosome labeling and uptake
The isolated exosomes were incubated with the anti-CD9 antibody for 1 h at 4°C, followed by incubation with Alexa Fluor-546 conjugated secondary antibody for 1 h at room temperature, protected from light.The unreacted antibodies were removed by ultracentrifugation.
Fluorescently labeled exosomes (FBM and SCC131) were co-cultivated with the recipient cells (MCF10A) for 3 h, in serum-free media.Cells were trypsinized and washed thrice with 1X PBS, and the exosomal uptake was analyzed using flow cytometry in BD FACSVerse (BD Biosciences, San Jose, CA, USA).The fluorescence count (PE-586/42 filter) was taken in the gated region.The gating region was chosen based on the reading from non-fluorescent control cells, and the graph was plotted with the percentage of positive cells in the gated region (10000 events/sample) for FBM-and SCC131-exosome treatment.The analysis was performed using BD FACSuite software (BD Biosciences).Uptake was confirmed by Zeiss Axio Observer Widefield inverted fluorescence microscope, at room temperature using 20X/0.5-NAobjective, and AxioCam MRm 3 (S/N 4283).Nuclei were stained with DAPI (blue), exosomes were fluorescently immunolabelled with Alexa Fluor-546 (red), and cell boundary was visualized in phase contrast.Image acquisition was performed in Zeiss Lite 2011 software, and the channels were merged together in ImageJ 1.52n.Exosomes were labeled with a fluorescent dye, Dil (1,1′dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate) (Thermo Scientific, Waltham, MA, USA). 1 μM of freshly prepared Dil dye was added to the exosomes (in 1X PBS) and incubated at room temperature for 1 h with gentle mixing.The exosomes were pelleted by ultracentrifugation (1,00,000 xg, for 1 h at 4 °C) to remove the excess dye, and their uptake [19,20] was studied in MCF10A cells.

EMT and Migration Assay
The epithelial-mesenchymal transition of MCF10A and T4074 cells was studied by coculturing with FBM and SCC131 exosomes for 24 h, and the levels of vimentin, N-cadherin, and E-cadherin were detected using western blotting.Transwell migration assay was performed on a 24-well plate with 8 μM pore-sized transwell inserts.5 x 10 5 cells were added in the above Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016chamber in serum-free media.50 μg of exosomes were added in the upper chamber, and serumfree media was added in the lower chamber.After 24 h of incubation, cells on the upper chamber were removed, and migrated cells on the lower surface of the transwell inserts were fixed and stained with the Hemacolor staining kit (Merck, Kenilworth, New Jersey, USA).Five random, microscopic fields were observed, and the migrated cells were counted.Brightfield images were taken in Zeiss Axio Observer Widefield inverted microscope, 10X/0.3-NAobjective, and AxioCam ERc 5s (S/N MKG5250) camera.Image acquisition was carried out using Zeiss Lite 2011 software.

Phosphatidylserine level in exosomes
25 μg of exosomes were resuspended in 100 μL of annexin V-binding buffer (BD Biosciences, San Jose, CA, USA) and centrifuged at 100000 xg to remove the unbound dye.The pelleted exosomes were washed twice and resuspended in 100 μL of 1X PBS, and the FITC fluorescence (λ ex -495 nm; λ em -510-650 nm) was noted.

Liposome Preparation
Lipids (DOPC, DOPS & DOPE) in chloroform were mixed in the desired molar ratio and dried under nitrogen gas followed by lyophilization.Dried lipids were hydrated in water for 60 -90 min, and unilamellar vesicles were prepared using polycarbonate membranes of 100 nm pore size as described [21].Protein and vesicles were incubated at neutral pH buffer (20 mM Tris pH 7, 50 mM NaCl) for 5 min, and spectral measurements were acquired.1 mM and 150 µM of liposomes were used for circular dichroism and fluorescence experiments.Each spectrum is an average representation of 60 scans.

Sequence Analysis
The primary sequence of calnuc retrieved from the UniProt database (Accession ID: Q02818) was employed for the bioinformatic analysis.The helical wheel was constructed using the online server, Heliquest (http://heliquest.ipmc.cnrs.fr/)[22].

Gene Transfection
Calnuc gene was cloned into the pEGFPN1 mammalian expression vector.Calnuc ΔCT (C-terminal deletion) was generated by site-directed mutagenesis.Plasmids encoding GFPtagged Calnuc FL and Calnuc ΔCT were transfected to the SCC131 cells using FuGENE Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016transfection reagent (Promega, Madison, WI, USA).Exosomal expression was analyzed using western blotting.

Expression and Purification of Calnuc and fragments
The human Calnuc gene was cloned in pTYB12 vector, and fragments were generated by inserting stop codon using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Ipswich, MA, USA).Expression and purification of full-length calnuc and its fragments were performed as previously described [23].
The full-length Drosophila calnuc cDNA (1.522 kB) was obtained from Drosophila Genomics Research Centre (DGRC, St. Bloomington, IN, USA).The near full-length DmCalnuc gene (encoding 22-464 amino acids) was cloned into a pGEX-6P-1 vector, expressed, and purified as described [24].The eluted chimera was cleaved using PreScission protease (25:1 ratio of protein: protease) in cleavage buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA) for 16 h at 4 ºC.Cleaved GST tag and GST-PreScission protease chimera were removed by loading into a new GST agarose column.The eluted protein was concentrated and stored at -80 ºC until further use.The purified protein was found to be 95% pure as analyzed using 12% SDS PAGE.

pH studies
Glycine-HCl (pH 2), Sodium Acetate (pH 4), Tris-HCl (pH 8), and  buffers were used at a concentration of 50 mM.Protein samples were equilibrated in the appropriate pH buffers for 12 h, at room temperature, and used for further experiments.

Circular Dichroism (CD) Measurements
CD experiments were performed on Jasco J-815 spectropolarimeter (Jasco Corporation, Tokyo, Japan) connected to the Peltier controller for maintaining temperature.All CD measurements were carried out at a scan rate of 100 nm/min, and an averaged spectrum of three scans was taken for analysis.Far-UV CD spectrum was recorded from 195-250 nm in a quartz cuvette of path length 1 mm.Near-UV CD spectrum was recorded from 250-320 nm in a 10 mm path length cuvette.Buffer baselines were subtracted from the respective samples.5 μM of calnuc was used for far-UV CD spectroscopy, while the near-UV CD was recorded with 32 μM of calnuc.Mean Residue Ellipticity [θ MRE ] was calculated as mentioned where θ ellipticity (mdeg), C is protein concentration (M), l is cuvette path length (cm), and nnumber of peptide bonds in the protein.The secondary structure content of calnuc was analyzed using the DICHROWEB, an online tool [25].

Fluorescence Spectroscopy
Fluorescence studies were carried out in Fluorolog Spectrofluorometer (Jobin Yvon, Horiba Scientific, Piscataway, NJ, USA) with slit widths of 5 nm for both excitation and emission.The protein fluorescence spectrum was recorded with an excitation wavelength of 295 nm, and the emission spectrum was collected from 310-440 nm.For 8-anilinonaphthalene-1sulfonic acid (ANS) binding studies, samples were excited at 365 nm, and the corresponding emission spectrum was recorded from 440-600 nm.Each spectrum was an average of three spectra with appropriate blank corrections.All fluorescence experiments were performed with a protein concentration of 500 nM at 25 °C.

Size Exclusion Chromatography (SEC)
In order to find the compactness of the protein native and molten globule states, its elution profiles in size exclusion chromatography were analyzed.9.2 μM of Calnuc (incubated in the appropriate pH) was loaded onto a Superdex 200 GL (GE Healthcare, Chicago, IL, USA) column pre-equilibrated with corresponding buffers.

Dynamic Light Scattering (DLS)
Hydrodynamic radii (R H ) of the calnuc, at pH 4 and pH 8, was calculated from Microtrac Particle Analyzer (Betatek Inc, Toronto, ON, Canada).9.2 μM of calnuc was used, and the size distribution profile was plotted.The mean value was used to represent the hydrodynamic radii (R H ) of the protein.The size of exosomes was analyzed using DLS as described [18].

Thermal stability studies
Thermal unfolding of calnuc at pH 4 and 8 was monitored by far-UV CD melting studies.
A scan rate of 1 C/min was used with 30 s hold time at each temperature.Melting temperature (T m ) was calculated using the Sigmoidal Boltzmann Fit from a plot between fraction unfolded (f U , with respect to θ 222 value) vs temperature (T).Differential scanning calorimetry (DSC) experiments were done using 25 μM calnuc in a MicroCal VP-DSC instrument (GE Healthcare, Chicago, IL, USA), and data were analyzed with MicroCal Origin software supplied by the manufacturer.For pH 8 melting studies, calnuc is extensively dialyzed against (buffer exchanged) 50 mM HEPES and 50 mM acetate for pH 8 & pH 4, respectively.Corresponding buffers were used in the reference cell for appropriate pH scans.Both protein and the buffer were degassed before loading.Buffer-buffer baselines were obtained until proper equilibration of the instrument.Cells were kept at a constant pressure of 28 psi with a scan rate of 60 C/h.The twostate model was used for fitting in order to obtain thermodynamic parameters ΔH m (melting enthalpy) and T m .

Pathophysiological significance of the calnuc expression
A preliminary report demonstrated the elevated secretion of calnuc in CT26 murine colon carcinomas [26].This prompted us to investigate its expression in relevant disease models.
Calnuc's expression was monitored in the pre-clinical model of colon carcinogenesis by immunostaining the colon tissue samples resected from azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated and untreated control mice groups (Fig. 1A).Calnuc expression was monitored in mice colon tissue, 31 and 90 days after treatment, a standard model to study colon carcinogenesis [17].Immunostaining revealed that calnuc expression was significantly high in the 31-day treated group (Fig. 1B) with the quantitative scores (Q scores) of untreated mice versus azoxymethane-treated mice as 1 & 5, respectively (p-value of 0.05).On the other hand, comparison of calnuc immunostaining in 90-day treated and untreated mice lead to Q-scores of 6.34 & 3.34, respectively (Fig. 1C).This data specifies that increased calnuc expression is an early indicator of carcinogenesis, as documented by its high expression in the 31-day carcinogen exposed mice colons.

Tumor-specific exosome packaging of calnuc
Conditioned media collected from FBM (non-tumor, fetal buccal mucosal cells) and SCC131 (human oral squamous cell carcinoma) cell lines were concentrated and analyzed for the secretory levels and exosomal incorporation of calnuc (Fig. 2AB).Western blot analysis revealed the presence of calnuc in total extracellular fractions of SCC131 at higher levels compared to FBM, while intracellular levels of calnuc remained unaltered, implying the increased secretion of Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016calnuc is tumor-specific.The extracellular fraction of calnuc showed a molecular mass of 55 kDa, while the intracellular fraction was found to be 63 kDa (Fig. 2C) in accordance with previous independent studies [27,28].Exosomes were isolated from conditioned media, and the presence of calnuc was confirmed by western blotting.DLS measurements validated the dimensions [29] (90-150 nm) of the isolated exosomes (Supplementary Fig. 1).Exosomemediated secretion of calnuc was highly specific to cancerous cells as it is observed only in SCC131-derived exosomes, not in FBM-derived exosomes (Fig. 2D).CD9, an exosomal marker, was used as a positive control.Similar tumor-specific exosomal secretion of calnuc was observed in pancreatic cell lines (HPDE6 and MiaPaCa2) (Supplementary Fig. 2), thereby reaffirming the tumor-specific exosomal packaging and the biomarker potential of calnuc.The presence of calnuc in the tumor-derived exosomes also indicates its vital role in tumor development/progression.However, further clinicopathological studies are essential to ascertain calnuc as a prognostic marker.

Uptake of calnuc-packaged exosomes
Exosomes isolated from FBM and SCC131 cell lines were labeled with the fluorescently conjugated antibody and co-cultured with the non-tumorigenic breast epithelial cells (MCF10A).
The uptake of exosomes was visualized under a fluorescence microscope.We identified that SCC131-exosomes (containing calnuc) were internalized in MCF10A cells in high number, while FBM-exosomes (devoid of calnuc) were sparingly internalized (Fig. 3A, B).The percentage of exosome uptake by the breast epithelial cells was quantified using flow cytometry, revealing that 78% of MCF10A cells were transformed upon co-culturing with calnuc-containing exosomes (SCC131), while only 20% of cells were positive for calnuc-devoid exosomes (FBMexosomes) (Fig. 3C).These results highlight the potential of calnuc-containing exosomes in intercellular communication.In order to rule out the non-specific uptake of labeled antibodies, we also labeled the exosomes using a red fluorescent dye, Dil.The intake of Dil-labelled exosomes by MCF10A cells also displayed similar results, which validated the increased uptake of calnuc-containing exosomes (Fig. 3D, E).Metastasis is marked by the upregulation of N-cadherin and vimentin accompanied by the downregulation of E-cadherin, leading to EMT and subsequent cell migration [30,31].We analyzed the levels of these EMT markers in the non-tumor cells upon treating them with the exosomes.Calnuc-packaged exosomes augmented the expression of vimentin and N-cadherin, while E-cadherin was downregulated in MCF10A cells (Fig. 4A).On the other hand, calnucdevoid exosomes did not show significant changes in the levels of EMT markers.Similar changes were observed when the exosomes were added to the primary oral keratinocyte cells (T4074) (Fig. 4A).Further, the migration potential of exosomes was studied using a transwell migration assay.Calnuc-containing exosomes showed seven-fold and four-fold increased migration in MCF10A and T4074 cells, respectively.No significant difference in transwell migration was elicited by calnuc-devoid exosomes and PBS treatment (Fig. 4B), thereby establishing the importance of calnuc-packaged exosomes in cellular migration.Our findings suggest that calnuc-containing exosomes are potential candidates in driving EMT and migration, leading to metastasis.

Phosphatidylserine (PS) binding plays a critical role in exosomal sorting
Phosphatidylserine (PS) forms the principal constituent of the exosomal membrane [32].
We used FITC-conjugated annexin V to analyze the levels of phosphatidylserine in exosomes.
To identify the putative PS-binding site on calnuc, we scanned for the presence of dibasic amino acid motif (KK/RR/KR/RK) alongside hydrophobic amino acids, a general outline of a PS-binding site in proteins [33,34].The identification of the PS-interacting region and increased level of calnuc in PS-rich exosomes suggest the role of PS in regulating exosomal incorporation of calnuc.The primary sequence analysis of calnuc (UniProt ID: Q02818) has RK/KR-rich motif in addition to the hydrophobic residues in its C-terminal region (240-461), suggesting it to be the PS-interaction site in calnuc.The helical wheel projection of calnuc displayed the amphipathic nature of the helix (formed by 347-364 residues of the heptad repeat) (Fig. 6A, B), which is Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ2021001613 associated with membrane binding [35].To validate this prediction, we overexpressed Calnuc FL and Calnuc ΔCT in SCC131 cells and analyzed their exosomal incorporation.The deletion of the PS-binding site (C-terminal region) disturbed the exosomal packaging of calnuc (Fig. 5B).
Furthermore, we used liposomes as an in vitro exosome-mimic for analyzing PS-calnuc interaction.Small unilamellar vesicles were prepared with varying concentrations of phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE).Calnuc adopted a highly helical conformation upon interaction in the presence of PS-containing liposomes, monitored by an increase in negative ellipticity in the far-UV CD region (Fig. 5C, Supplementary Table 1).Additionally, a complete loss in the tertiary structural signature of calnuc is elicited upon interaction with PS-liposomes, indicating the formation of a molten globule conformation, which is described in detail in the subsequent sections (Fig. 5D).A defined secondary structure, with a total/significant loss in tertiary contacts, is the characteristic feature of the molten globule state in proteins [36].
Tryptophan residues were shielded from the polar environment (~7 nm blue shift accompanied by a marginal increase in fluorescence intensity) upon binding to PS-liposomes as analyzed from our intrinsic fluorescence measurements (Fig. 5E).Conformational changes in calnuc increased with an increase in the PS concentration in the liposomes, while no structural/conformational changes were observed in liposomes lacking phosphatidylserine (Fig. 5E).

C-terminal region of calnuc interacts with phosphatidylserine
The predicted PS-interaction site in calnuc was further validated by analyzing the liposome-mediated secondary structural changes using deletion constructs.Spectroscopic analysis with the N-and C-terminal fragments of calnuc revealed that the C-terminal region of calnuc interacts with PS-liposomes, demonstrating an increased secondary structural content and fluorescence intensity.The N-terminal fragment did not show any changes in secondary structure or fluorescence intensity, thereby confirming our prediction (Fig. 6C-F).Thus, the role of the Cterminal region in the structural plasticity of the protein towards membrane interaction/association is highlighted.Acidic phospholipids (like phosphatidylserine) lower the microenvironmental pH, which causes structural perturbances in proteins, leading to the molten globule conformation [37].

Acidic pH induces a molten globule state in calnuc
Hence, we monitored the pH-mediated structural/conformational changes in calnuc using various biophysical techniques.Calnuc has two tryptophan residues (at 232 and 333 positions) located before and after the EF-hand domain.The fluorescence spectrum of calnuc at pH 1-12 is presented in Supplementary Fig. 3A.Tryptophan fluorescence of human recombinant calnuc at pH 8 (native state), with λ max, em at 352 nm, indicates a complete exposure of these residues to the aqueous environment.At pH 4, a blue shift of ~ 7 nm (λ max, em at 345 nm) was accompanied by an increase in emission intensity, implying movement of the tryptophan residues to a more hydrophobic region (Fig. 7A).The fact that calnuc presented a unique change in secondary and tertiary structure at pH 4 was further explored (Supplementary Fig. 3B, C).At pH 4, a slight increase in the helical content of calnuc was observed compared to its native state (Fig 7B, Supplementary Table 1).Fig. 7C represents the near-UV CD spectrum of human calnuc that demonstrated a remarkable loss in its tertiary structure at pH 4 compared to pH 8. A change in fluorescence intensity of protein-bound ANS is considered a distinguishing feature of the molten globule conformation of proteins [38].A six-fold increase in ANS fluorescence emission was observed upon binding to calnuc at pH 4 compared to pH 8 (native state), indicating a relative increase in exposed hydrophobic residues (Supplementary Fig. 4A).
DmCalnuc elicited fluorescence emission λ max, em similar to its human homolog (~ 354 nm) at pH 8.By lowering the pH to 4, we noticed a 7 nm blue shift in fluorescence emission maximum accompanied by a minimal drop in intensity at pH 4 (Fig. 7D).The binding of ANS to DmCalnuc also led to a five-fold increase in its intrinsic fluorescence at pH 4 compared to pH 8 (Supplementary Fig. 4B).Far-UV CD spectral signatures of DmCalnuc (exhibits 45% sequence identity) at pH 8 and pH 4 presented a considerable rise in helicity at pH 4. (Fig. 7E, Supplementary Table 1).Fig. 7F represents a total loss in the structural fold of DmCalnuc at pH 4 compared to pH 8, like human calnuc.These results confirm the formation of acidic pHinduced molten globule structure in human and Drosophila calnuc, mimicking the membranebound state.A change in the hydrodynamic radius (R H ) is generally observed in a protein upon adopting a molten globule state that can be monitored by size exclusion chromatography and dynamic light scattering.Fig. 8A reveals the elution profiles (size exclusion) of calnuc at pH 4 & pH 8.At pH 8, a mixture of dimer and monomeric calnuc was observed, with a substantially larger dimeric fraction.The dimeric nature of calnuc (at pH 8) was in accordance with our native page analysis (data not shown) and an earlier report [39].At pH 4, a significant increase in the monomer population indicated the monomeric nature of molten globule calnuc (Fig. 8A inset).A shift in the oligomeric profile of calnuc during molten globule formation (dimer to monomer) was further confirmed by our DLS analysis (Fig. 8A inset table, Supplementary Fig. 4A, B).

Molten globule calnuc is a monomer
Since molten globule calnuc was identified as a monomer, we hypothesize that calnuc exists as a monomer in PS-liposomes and PS-rich exosomes..

Decreased thermostability of molten globule state aids in the exosome-mediated secretion
The thermostability of the molten globule calnuc was characterized by temperaturedependent far-UV CD spectroscopy and differential scanning calorimetry (DSC).A plot of the fractional population of unfolded species obtained from ellipticity values at θ 222 , vs temperature (T), illustrates a broad transition.Melting temperature (T m ) of native calnuc (pH 8) was found to be 50 °C, whilea change in pH to 4 (MG state) reduced its T m to 40 °C (Fig. 8B, Supplementary Table 2).The loss of tertiary structural elements at pH 4, probably leading to the decreased stability of the molten globule state (as indicated by far-UV CD signature).Results from our DSC studies (Fig. 8C and Supplementary Table 1) also confirmed the reduced thermodynamic stability of calnuc at pH 4, a characteristic hallmark feature of the molten globule state.

DISCUSSION
Calnuc, a DNA-and calcium-binding protein, is involved in the pathophysiology of various cancers [10].Expression levels of calnuc are significantly higher in non-Hodgkin's lymphoma patients and gastric adenocarcinoma-associated lymph node metastasis patients, emphasizing its role in metastasis [6][7][8].Calnuc secretion is reported in the pituitary and colon cancer cell lines and in the sera of autoimmune mice, recognizing it as a prospective cancer biomarker [6,9].However, these reports fail to address the mechanism underlying its tumorcentric secretion.Our work illustrates how the increased expression of calnuc is related to the Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016etiology of cancers.The elevated expression of calnuc in our colon carcinogenesis model (Fig. 1B,C) corroborates with the earlier reports [6,7,26] and substantiates it as a promising biomarker in early cancer diagnosis.Several cancer biomarkers (KRAS, c-Myc, and TGFβ) were upregulated in the AOM/DSS-induced colons supported the plausibility of calnuc as a prognostic cancer biomarker [17,40,41].
The cancer secretome comprises a repertoire of tumor biomarkers [2], including exosomal proteins that are vital for cancer diagnosis and therapy [16].Exosomes not only transport physiological signals (stemness, immune response) [42] but are also involved in shuttling oncogenic factors (proteins and miRNA) to the surrounding non-tumor cells leading to drug resistance, tumor progression, invasion, angiogenesis, and metastasis [29].Exosomes are vesicles of endosomal origin [32], and the endosomal localization of calnuc provided insight towards studying its exosomal sorting [43,44].Like calnuc, several oncogenic proteins (KRAS, CD44, EGFR, H-Ras, KRAS, MDM2, and KIT) that are instrumental for cancer proliferation are upregulated in the tumor exosomes [45,46].Additionally, the "Vesiclepedia" database reports the presence of calnuc in the exosomes isolated from different tumors (astrocytoma, breast cancer, colorectal cancer, kidney cancer, leukemia, melanoma, ovarian carcinoma, and prostate cancer) [47], signifying its importance in cancer pathophysiology.Thus, the tumor cell-specific exosomal packaging of calnuc illustrated in the current study highlights its potential as a prognostic cancer biomarker (Fig. 2D).Calnuc-containing exosomes triggered tumorigenic properties in the recipient non-tumor cells, including EMT and migration (Fig. 4), which are the established physiological events reported upon calnuc expression [48], thereby identifying its role in tumorigenesis and metastasis-inducing machinery.This primary evidence on exosomal calnuc as a component metastatic-inducing machinery, if extended to exosomes from clinical and animal models, could delineate its potential is molecular oncology.
We further investigated the possible mechanistic basis of tumor-specific exosomal packaging of calnuc.Elevated levels of phosphatidylserine (PS) have been reported in ovarian cancer [14], and PS-positive exosomes are acknowledged as a diagnostic marker for early-stage malignancies [49].PS-binding motif in a protein comprises positively charged amino acids and hydrophobic residues, which facilitate electrostatic interactions and lipid anchorage, respectively distribution is identified in the C-terminal region of calnuc that acts as a potential PS-interaction site (Figure 5A,B).This putative PS-binding region of calnuc comprises a heptad repeat of leucine residues (leucine zipper, 347-403), forming a coiled-coil conformation [48,56] as observed in other zipper proteins.Such heptad leucine repeats are suggested to form the membrane-spanning interface in phospholamban [57] and M2 proton channel [58].Gurezka et al. (1999) illustrated the self-assembly of the monomeric heptad leucine motif in the artificial membranes [59].The absence of structural information of calnuc is a huge setback in understanding the intricacies of its structure and function.Hence, preliminary structural modeling in calnuc [48] yielded a helix-turn-helix structure in the C-terminal region similar to the PS-binding region observed in PS receptor-1 [60].Along with our prediction analysis (Fig. 6A,B), in vivo (Fig. 5A,B) and in vitro (Fig. 5C-E, Fig. 6C-E) experiments established the importance of calnuc-PS interaction in exosomal incorporation or vesicular assembly.Analogous to this observation, PS-dependent exosomal or vesicular packaging has been demonstrated in lactadherin (a calcium-binding protein) [61,62], proteinase 3 (PR3) [63,64], PDK1 [54], hsc-70 [55], and gas6 [65].Apart from this, PS-protein interaction is well established as a regulator of protein targeting to the cell membrane, secretory vesicles, endosomes, and lysosomes [54,66,67].
Thus, identifying the mechanism underlying the exosomal packaging of calnuc opens up a new possibility in targeted drug design for cancer.
Additional support for our proposed mechanism comes from a detailed study of the interaction of calnuc with PS involving a switch in the protein's conformation.
Phosphatidylserine perturbs protein conformation (a loss in tertiary fold and exposure of hydrophobic residues) by generating an acidic microenvironment, forming a molten globule state [37,[68][69][70].Similar conformational changes are elicited in vitro in calnuc at acidic pH (pH 4), demonstrating its responsiveness towards the low pH environment in cancer cells and driving molten globule conformation (Figure 7A-C) [71].The PS-interacting C-terminal region of calnuc was largely unstructured as per our prediction analysis, which indicates the molten globule propensity (Supplementary Fig. 5).These observations enable us to advocate that a conformational switch (MG state formation) in a protein is essential in facilitating its membrane association and exosomal packaging (Fig. 5B).It is noteworthy that limited literature exists on the occurrence of 'molten globule' conformation in multi-domain proteins of the size range of calnuc (Supplementary Table 2).Since molten globule calnuc is a monomer (Figure 8A), we hypothesize the presence of monomeric calnuc in PS-rich exosomes.A similar molten globuleinduced dissociation of the dimeric state has also been illustrated in yeast glutathione reductase and human arc repressor [72,73].The fact that molten globule state is vital for membrane association and leading to its secretion is discussed in detail in various proteins (bacterial toxins Therefore, our mechanistic model highlights the interaction of the C-terminal region of calnuc with phosphatidylserine lipids in the cancer cell.This interaction initiates molten globule conformation by altering the microenvironmental pH, leading to the subsequent exosomal packaging and secretion.Calnuc-packaged exosomes are taken up by non-tumor recipient cells and elicit tumorigenic properties like migration and EMT (Fig. 9).The fact that calnuc-devoid exosomes did not elicit a similar metastatic effect further reinforces our model.This work provides a basis for exploiting calnuc as a critical candidate for drug target in cancer treatment.

Figure 1 .
Figure 1.Immunostaining for calnuc in mice colon tissue sections.(A) A schematic representation of the protocol followed as described [17].(B) Expression levels of calnuc in AOM-DSS-injected mice colon tissue sections (31-and 90-day treatment group), along with a healthy/untreated control group for comparison.Calnuc expression was high in the mucosal layer of the AOM-DSS-induced colitis group, as compared to low expression in the untreated group.(C) Calnuc expression in the tissue sections was quantified using the Q-score, and the values were plotted.Scale bar, 100 μm.(n = 3, mean ± SE, *p<0.1).

Figure 2 .
Figure 2. Total-and exosomal-secretion of calnuc in normal and tumor cell lines.(A) Schematic representation of the extraction/collection of exosomes and total secreted protein from cell lines.(B) Representative figure of a typical exosome and its contents.(C) Western blot illustrating the expression levels of calnuc from the intracellular and total extracellular fraction of non-tumor (FBM) and cancer cell line (SCC131) after incubating the cells in the conditioned media for 24 h.(D) Intracellular and exosomal expression of calnuc in FBM (non-tumor) andSCC131 (head and neck cancer).Exosomes were isolated from the conditioned media of these cell lines, and the presence of calnuc and CD9 (an exosomal marker) were detected using western blotting.Cytochrome C, a negative marker for exosomes, was also used.Experiments were performed in triplicates, and the representative image is shown.

Figure 4 .
Figure 4. EMT induction and migration properties of calnuc-packaged exosomes.(A) Expression levels of EMT markers (N-cadherin, E-cadherin, and vimentin) in MCF10A and T4074 cells were monitored by western blotting upon treatment with 1X PBS (control), calnucdevoid exosomes (FBM-exosomes), and calnuc-containing exosomes (SCC131-exosomes).Blots were cut into desired molecular mass sections before incubation with primary antibodies and treated individually.Experiments were performed in triplicates, and the representative image is shown.The panel at right shows the densitometric quantification of the data (n = 3, mean ± SE, ***p<0.001).(B) Representative bright-field images of exosome-induced transwell migration in MCF10A and T4074 cell lines upon treatment with 1X PBS (control), calnuc-devoid exosomes (FBM-exosomes) and calnuc-containing exosomes (SCC131-exosomes).Cells were stained with the Hemacolor kit (Merck, Kenilworth, New Jersey, USA), and the number of cells that were directly transmigrated across the porous membrane after 24 of treatment was counted using a bright-field microscope.Scale bar, 5 μm.The panel at right shows a quantitative analysis of the data (n = 4; values represented mean ± SE, ****p<0.0001).

Figure 5 .Figure 8 .
Figure 5. Interaction with phosphatidylserine-containing liposomes initiates molten globule conformation in calnuc.(A) Fluorescence intensity of the annexin V-FITC conjugate bound to exosomes corresponds to PS levels in exosome membrane (n = 3, mean ± SE, ***p<0.001).(B) Western blotting analysis of intracellular fractions and exosomes of SCC131 cells overexpressing EGFP-tagged calnuc FL and ΔCT.Expression of GFP chimeras and CD9 (exosomal marker) was detected by western analysis.(C) Secondary-and (D) tertiary-structural changes in calnuc monitored using far-UV CD and near-UV CD spectroscopy, respectively, in

Figure 9 .
Figure 9. Exosomal calnuc is a tumor biomarker and metastasis inducer.Calnuc adopts a molten-globule structure upon interaction with phosphatidylserine.Exosomes are PS-rich vesicles and the interaction of calnuc with phosphatidylserine favor exosomal-packaging.Calnuc-packaged exosomes are likely to be transported through the blood and other body fluids and are internalized in other cells, thereby driving EMT and migration, leading to metastasis and tumor progression.This postulates calnuc as a potential metastasis inducer and tumor biomarker.
Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016

Table 3 .
com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 a -Far-UV CD melting, b -DSC Thermodynamic parameters for molten globule and native state of calnuc.Sigmoidal fit is used in calculate T m in Graphpad Prism 6 software.In the case of DSC, the two-state model available from Microcal software (provided by the manufacturer) is used to find the melting temperature (T m ) and calorimetric enthalpy of unfolding (ΔH cal ).Molten globule conformation in diverse proteins."Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210016/912976/bcj-2021-0016.pdf by guest on 05 June 2021 Biochemical Journal.This is an Accepted Manuscript.You are encouraged to use the Version of Record that, when published, will replace this version.The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210016 "Supplementary