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A complex dance: the importance of glycosaminoglycans and zinc in the aggregation of human prolactin Line Friis Bakmann Christensen, Kirsten Gade Malmos, Gunna Christiansen, and Daniel Erik Otzen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00153 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 16, 2016

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Biochemistry

A complex dance: the importance of glycosaminoglycans and zinc in the aggregation of human prolactin Line Friis Bakmann Christensen1, Kirsten Gade Malmos1, Gunna Christiansen2 and Daniel Erik Otzen*1 1Center

for Insoluble Protein Structures, Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, Aarhus University, 8000 Aarhus C, Denmark

2Department

of Biomedicine-Medical Microbiology and Immunology, Aarhus University, 8000 Aarhus C, Denmark

*Author to whom correspondence should be addressed. Center for Insoluble Protein Structures, Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, Aarhus University, 8000 Aarhus C, Denmark. Phone: +45 87156741. E-mail: [email protected].

1 ACS Paragon Plus Environment

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Abbreviations ANS, 8-anilino-1-naphthalenesulfonic acid; CBB, Coomassie Brilliant Blue; CD, circular dichroism; CSA, chondroitin sulfate A (CAS Number: 39455-18-0); EDTA, ethylene-diamine-tetra-acetic acid; ER, endoplasmic reticulum; FTIR, Fourier transform infrared; GAGs, glycosaminoglycans; HEPES, 4-(2-hydroxyethyl)-piperazine-1ethanesulfonic acid; hPRL, human prolactin; HS, heparan sulfate; IPTG, isopropyl β-D-thiogalactopyranoside; ITC, isothermal titration calorimetry; MES, 2-(N-morpholino)-ethanesulfonic acid; PMSF, phenyl-methyl-sulfonyl fluoride; PRL, prolactin; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; TGN, transGolgi-network


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Abstract The zinc-binding hormone pituitary human prolactin (hPRL) is stored in secretory granules of specialized cells in an aggregated form. Glycosaminoglycans (GAGs) are anionic polysaccharides commonly associated with secretory granules, indicating their involvement in granule formation. Here we, for the first time, study the impact of GAGs in combination with Zn2+ on the reversible hPRL aggregation across the pH 7.4-5.5 range. Zn2+ alone causes hPRL aggregation at pH 7.4 while aggregation between pH 7.4 and 5.5 requires both Zn2+ and GAGs. GAGs alone cause hPRL aggregation below pH 5.5. Comprehensive thermal stability investigations show that hPRL is particularly destabilized towards thermal denaturation at pH 5.5 and that GAGs increasingly destabilize hPRL at decreasing pH. We propose that Zn2+ causes hPRL aggregation through low-affinity Zn2+ binding sites on hPRL with GAGs facilitating Zn2+ binding by neutralizing repulsive positive charges of hPRL in the acidic environments of the TGN and mature secretory granules. Independent of the aggregation-causing agent(s), the different hPRL aggregates show very similar secondary structure and amorphous morphology. We speculate that this may be a recognizable sorting signal in the formation of hPRL granular vesicles.


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Human prolactin (hPRL) is a hormone produced in tissues throughout the human body, but is only stored in secretory granules in the lactotroph cells of the anterior pituitary. Storage in these specialized cells allows bursts of hPRL to be released by exocytosis, resulting in an continuous increase in serum levels of hPRL during pregnancy (1, 2). Prolactin (PRL) was first isolated from cows and received its name due to its ability to induce lactation in guinea pigs, rabbits and monkeys (3). Subsequently, more than 300 biological functions have been assigned to hPRL (4, 5). Pituitary hPRL is synthesized as a 227 amino acids long pre-prolactin encoding both the protein and a 28 amino acids long signal peptide directing the transport across the endoplasmic reticulum (ER) membrane (6). The folded hPRL moves through the secretory pathway, where it encounters increasingly acidic environments; the pH in the trans-Golgi network (TGN) is close to 6.0 and can reach 5.5 in the final, mature granules (7). Aggregation of PRL is initiated along the Golgi concave trans face (8) and the dense cores of the granules are formed by budding off of small vesicles around the aggregates after fragmentation of the TGN (9). Studies in AtT20 cells have shown that the acidification through the secretory pathway is important for hPRL aggregation (10). High concentrations of Zn2+ in the secretory pathway of neuroendocrine cells (11) and the ability of Zn2+ to cause hPRL aggregation at neutral pH (10, 12) suggest an important role of Zn2+ as well. However, aggregation of hPRL by Zn2+ is independent of the high-affinity Zn2+ binding site of hPRL which involves the histidine at position 27 (12). The glycosaminoglycans (GAGs) heparan sulfate (HS) and chondroitin sulfate A (CSA) (13, 14) are found in PRL granules. GAGs are anionic polysaccharides characterized on the basis of their core disaccharide unit which normally consists of a hexuronic acid and a hexosamine. They are further differentiated by their degree of sulfation: with three sulfate groups per disaccharide, heparin is the most sulfated GAG. The presence of GAGs in PRL granules is one of several examples of GAGs’ role in the formation of secretory granules (15), suggesting a common sorting of secretory proteins which involves GAGs. GAGs are also implicated in the fibrillation of numerous peptide hormones, and the fibril state has been proposed to be a natural storage form for hormones (16, 17). Furthermore, hPRL and heparin can interact directly, as hPRL can be retained on a heparin column (18). Here we investigate the role of Zn2+ and the GAGs CSA and heparin in hPRL aggregation. Over the pH range that hPRL experiences in the secretory pathway, GAGs and Zn2+ engage in an elaborate and role-shifting courtship with the protein, revealing a complex interaction system that can be tuned to lead to stably packaged aggregates. We show that reversible aggregation can be followed over time and completed within the timeframes of the processes of hPRL aggregation, packaging and solubilization in vivo. This is crucial to the biological relevance of the processes and an issue that is often overlooked in the formation of hPRL amyloid fibrils.


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Materials and methods Materials: The prolactin pT7-hPRL vector was a generous gift from Birthe B. Kragelund, University of Copenhagen (19). GAG molar concentrations were calculated on the basis of the disaccharide units glucuronic acid-[1→3]-NAcetylgalactosamine-4S (MW = 503.34 g/mol) for CSA sodium salt from bovine trachea and uronic acid-2S[1→4]-Glucosamine-NS-6S (MW = 665.40 g/mol) for heparin sodium salt from porcine intestinal mucosa. Both GAGs were purchased from Sigma-Aldrich (Munich, Germany). ZnSO4 was purchased from Merck Millipore (Darmstadt, Germany). All other chemicals were purchased from Sigma-Aldrich (Munich, Germany). Expression and purification of hPRL: Recombinant hPRL from the pT7-hPRL vector was grown in Escherichia coli BL21(DE3) cells on LB-agar plates (100 µg/mL ampicillin) and all colonies were transferred to and expressed in LB-media (100 µg/mL carbinicillin). Purification was performed as described (19, 20) with minor modifications. After reaching an OD600 of 0.6-0.8, protein expression was induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). Inclusion bodies were harvested by centrifugation and 1 mM phenyl-methyl-sulfonyl fluoride (PMSF) was added before cells were disrupted by sonication (3*3 min, 40% amplitude). Inclusion bodies were resuspended in 5 M guanidine hydrochloride (GuHCl), 15 mM β-mercaptoethanol and 20 mM sodium phosphate, pH 7.0, and incubated at 55˚C for 10 minutes to ensure reduction and unfolding of hPRL before returning to room temperature. To ensure proper refolding, hPRL was diluted to below 0.1 mg/mL and refolded by continuous dialysis against refolding buffer (20 mM NH4HCO3, 200 mM NaCl, pH 8.0) for three days, during which new refolding buffer was added at 2 mL/min to the bottom of the dialysis container. Subsequently, hPRL was exposed to simple dialysis for one day. Refolded protein was centrifuged, filtered (0.2 µM) and ~ 15-fold concentrated in a 350 mL Stirred Cell (MWCO 10 kDa ultrafiltration discs, Merck Millipore, Germany). Purification was done with size exclusion chromatography on a HiLoad™ Superdex™ 75 column (GE Healthcare, Buckinghamshire, England) in 20 mM NH4HCO3, 100 mM NaCl (pH 8.0) followed by ion exchange on a Sepharose HiTrap Q FF (GE Healthcare, Buckinghamshire, England) in 20 mM Tris-HCl (pH 8.0). Both columns were run with ÄKTA systems (GE Healthcare, Sweden). Monomeric protein was eluted with a NaCl gradient from 0-0.5 M and desalted against 25 mM HEPES/MES, 120 mM KCl (pH 7.4/6.0) before concentration to ~ 2 mg/mL in a 10 mL Stirred Cell (MWCO 10 kDa, Merck Millipore, Germany). The final concentration was determined with absorbance spectroscopy A280 nm on a NanoDrop1000 (Thermo Scientific, USA) using MW = 22.9 kDa and the calculated ε = 21,805 M-1cm-1 (21). Samples were stored at -20˚C.


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Thiol quantitation assay: The Measure-IT™ Thiol Assay Kit (Thermo Fisher, USA) was used to check for free thiols after refolding. The kit supplies assay reagent, dilution buffer and concentrated thiol standards (reduced glutathione) and has a linear range of 0.05-5 µM thiol. The Measure iT standards were loaded into wells of a NUNC-96 well plate (catalog #152037, Thermo Scientific, USA) as described in the manufacturer’s protocol. Two different hPRL concentrations of 0.36 µM and 0.72 µM (corresponding to cysteine concentrations of 2.1 µM and 4.3 µM, respectively, due to mature hPRL’s 6 cysteine residues at positions 32, 39, 86, 202, 219 and 227) were compared to the standards. Fluorescence was measured with excitation at 485 nm and emission at 520 nm using a TECAN GENios Pro Plate reader (Tecan, Switzerland). All samples were done in triplicates. ANS fluorescence: 8.7 µM hPRL was incubated 1 hour with 40 µM ANS and increasing CSAdisaccharide concentrations at pH 7.4 before adding 40 µM Zn2+. After 10 minutes of additional incubation, ANS fluorescence was measured on an LS55 luminescence spectrophotometer (Perkin Elmer Instruments, USA) with excitation at 365 nm and 400-600 nm emission. The experiments were performed in a black 3*10 mm quartz cuvette at 25˚C with slit widths of 10 nm and a scan speed of 100 nm/min. The average of three accumulations is shown. ThT fluorescence: 8.7 µM hPRL was incubated at room temperature for 1 hour with either 40 µM Zn2+ (pH 7.4) or 40 µM Zn2+ + 30 µM heparin (in units of GAG disaccharide concentration) (pH 6.0 and 5.5). 40 µM ThT was added and the samples were incubated for an additional 10 min before measuring ThT fluorescence at 25˚C. The measurements were performed on an LS55 luminescence spectrophotometer (Perkin Elmer Instruments, USA) with excitation at 450 nm and 460-550 nm emission. Slit widths were 10 nm, scan speed was 100 nm/min and three accumulations were averaged. A black 3*10 mm quartz cuvette was used. Circular dichroism spectroscopy: Far-UV CD spectra, aggregation kinetics and thermal denaturation measurements were recorded on a Jasco Spectropolarimeter (model J-810-150S, Japan) using a 1 mm quartz cuvette with a lid. Temperature was controlled by a Peltier element. The protein was diluted into different buffers: 25 mM sodium acetate, 120 mM KCl (pH 4.0/5.0), 25 mM MES, 120 mM KCl (pH 5.5/6.0), 25 mM HEPES, 120 mM KCl (pH 7.0/7.4) or 25 mM sodium borate, 120 mM KCl (pH 9.0) to a final concentration of 8.7 µM. Far-UV spectra were recorded at 250-190 nm with 0.1 nm intervals, scan speed of 50 nm/min and 4 s response. 5 repeat scans were averaged. All spectra were baseline corrected with respect to buffer. Thermal denaturation was carried out from 20˚C to 95˚C with a temperature slope of 1˚C/min at 222 nm, with a 4 s response, 0.1˚C data pitch and 2 nm band width, after which the scan was repeated in reverse. Before each thermal scan, 8.7 µM hPRL was


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incubated at the different pH-values either alone or in saturating molar ratios of 1:45 (hPRL to CSAdisaccharide) or 1:35 (hPRL to heparindisaccharide) for 15 min. Thermal unfolding/refolding of hPRL was fitted to a two-state transition by the Gibbs-Helmholtz equation previously described (22), using KaleidaGraph version 4.0 (Synergy Software, USA) to obtain values for the melting temperature, Tm. The error of fitting was not allowed to exceed 0.1˚C. A denaturation baseline was not always reached due to high protein stability and in these cases the denaturation baseline slope was omitted from the equation. Aggregation/disaggregation kinetics were followed as the ellipticity at 210 nm immediately after addition of Zn2+ or GAGs to monomeric or aggregated hPRL as described in the following table: pH 7.4 6.0/5.5

Aggregation (addition to monomeric hPRL) (1) Addition of Zn2+ (2) Pre-incubation with heparin, addition of Zn2+ (1) Pre-incubation with heparin, addition of Zn2+ (2) Pre-incubation with Zn2+, addition of heparin

Disaggregation (addition to pre-formed aggregates) (1) Addition of heparin (2) Addition of EDTA (1) Addition of EDTA

Measurements were performed at 10˚C until a stable baseline was reached. The data pitch was 2 s, response was 8 s and band width was 5 nm. Buffers were measured for 5 min and the average value was subtracted from every measurement before fitting the curves to either a single or a double exponential equation to get estimates of the rate constant, k. Isothermal titration calorimetry (ITC): ITC measurements were conducted at 10˚C on a VP-ITC calorimeter (MicroCal, Inc., USA). This temperature was chosen to allow direct comparison with CD data (also recorded at 10˚C). ZnSO4 and heparin were solubilized in the same buffer stock solutions (120 mM KCl and buffers indicated above) to minimize artifacts due to differences in buffer composition. All samples were thoroughly degassed before loading into the sample cell (1.5 mM heparin in disaccharide units) and the syringe (30 mM ZnSO4). Parameters were optimized to obtain a full baseline at high [Zn2+]/[heparindisaccharide] molar ratios with 5 µL injections and 300 s spacing. Zn2+ was also titrated into pure buffer under the same conditions to determine the heats of dilution. The heat signals were integrated using the Origin software (MicroCal, Inc., USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): To estimate pellet fraction, supernatants and pellets were mixed with 6X reducing loading buffer (G Biosciences, USA), heated to 90˚C and centrifuged


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shortly. 8 µL of the samples were loaded with a protein ladder (PageRuler™ Plus Prestained, Thermo Scientific, Lithuania) and three known concentrations of hPRL (2.2; 4.4 and 8.7 µM). 15% bis-tris SDS-polyacrylamide gels were used and run in bis-tris running buffer at 140 V for one hour. Gels were stained with Coomassie Brilliant Blue (CBB) (1.2 mM CBB, 5% ethanol, 7% acetic acid) for 20 min and de-stained overnight in a solution of 5% ethanol and 5% acetic acid. Gel Analyzer 2010a (23) was used to quantify all bands and the pellet fraction was calculated. Dilutions were accounted for in the calculations. Fourier transform infrared (FTIR) spectroscopy: Aggregates were formed by the addition of either Zn2+ (pH 7.4) or GAGs alone (pH 4.0 and 5.0) or the combination of the two (pH 5.5 and 6.0). The structure of the aggregates was investigated by carefully drying 2.0 µL of aggregated hPRL (obtained by aggregating 8.7 µM hPRL in 150 µL buffer, centrifuging and resuspending the pellet in 15 µL buffer) onto the crystal with nitrogen on a Tensor 27 FTIR (Bruker Optics, Billerica, MA). The software OPUS version 5.5 (http://www.stsci.edu/software/OPUS/kona2.html) was used to process the data, including calculating atmospheric compensation, baseline subtraction and second derivative analysis. All spectra are accumulations of 68 scans with a resolution of 2 cm-1 in the 1000-3998 cm-1 range. Only the 1520-1700 cm-1 range, comprising information about secondary structure, is shown. Transmission electron microscopy (TEM): 5 µL of the same samples as used for FTIR investigations were applied to a 400-mesh carbon-coated glow-discharged Ni grid. Grids were stained after 30 s with 1% phosphotungstic acid (pH 7.0) and blotted dry on filter paper. Samples were viewed in a transmission electron microscope (JEM-1010; JEOL) operating at 60 kV. Images were obtained using an Olympus KeenViewG2 camera. Results Purification of hPRL and its interactions with heparin at neutral pH: To address proper refolding of hPRL, we used a fluorescence-based Thiol Assay Kit used to confirm that all 6 cysteines present in the mature peptide form internal disulfide bonds so that no free cysteines are present. The standard curve fitted to a linear equation giving: y = 438±98 + 4712±37 * [free cysteine], R2 = 0.99 by linear regression (Fig. S1A). The two different protein concentrations gave signal values very close to the intercept value (y = 378±6 and y = 402±5, respectively), indicating zero signal within error. In addition, far-UV CD spectra of the refolded protein confirmed the secondary structure expected from the hPRL solution structure (Fig S1B) (19). To investigate potential differences in binding to hPRL between CSA and heparin, complexes were attempted formed and investigated with size exclusion chromatography (SEC). SEC chromatograms showed that hPRL was able to form a separately eluting complex


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with heparin but not CSA at pH 7.0 (Fig. 1A). Remarkably, increasing the pH to 7.4 essentially abolished complex formation (Fig. 1B). The interaction between heparin and hPRL at pH 7.0, however, did not have any major effect on hPRL stability. Thermal denaturation scans showed that hPRL alone had a Tm of 74.6˚C±0.1˚C and decreased slightly to 74.1˚C±0.1˚C with heparin added and to 73.9±0.1˚C with CSA added (Fig. 1C). Zn2+-induced hPRL conformational changes are counteracted by GAGs: The high concentration of Zn2+ in the secretory pathway (11) prompted us to investigate interactions between hPRL and Zn2+. With a pI of 6.2 (24), hPRL is negatively charged at neutral pH and able to interact with the positively charged zinc ions. These interactions make hPRL aggregate at pH 7.5 both in vivo and in vitro (10), and the structural consequences of this aggregation were investigated with ANS and circular dichroism (CD). At pH 7.4, hPRL alone gave a low ANS fluorescence signal at 465 nm, indicating that no hydrophobic areas are accessible to ANS in the natively folded protein (Fig. 2A), and the CD spectrum confirmed the α-helical content of native hPRL with minima at 222 nm and 209 nm (Fig. 2B). However, addition of Zn2+ increased the ANS signal intensity almost 4-fold (Fig. 2A) and the CD spectrum changed to a single 227 nm minimum (Fig. 2B). This unusual CD signal, which is not found in conventional α-helix or β-sheet structures, is not an artifact of light scattering from aggregates, since sonication to reduce aggregate size does not alter the spectrum (data not shown). These results confirm that the addition of Zn2+ changes the conformation of hPRL at pH 7.4, consistent with previous results (25). Remarkably, preincubation of hPRL with CSA suppressed the Zn2+-induced changes in a concentration-dependent manner according to both ANS fluorescence and CD (Fig. 2A/B). The titration curve in Fig. 2A could not be fitted satisfactorily to a single binding step. Rather, the data indicated that CSA bound in two steps, namely a first relatively high-affinity binding step complete around 40-60 µM CSA (dissociation constant estimated to ~8 µM, see text of Fig. 2A and associated fit) followed by a second step that was not complete within the measured concentration range, but has a dissociation constant of at least 300 µM. Heparin has a similar effect (Fig. S2). We confirmed the ability of GAGs to keep hPRL solubilized using SDS-PAGE to estimate the amount of hPRL in supernatant and pellet after centrifugation. Both GAGs inhibit aggregation in a concentration-dependent manner; heparin being slightly more effective than CSA (Fig. 2C). From SDS-PAGE it is also clear that the structures giving rise to the 227 nm spectral feature is in fact directly correlated to precipitable aggregates – therefore this is believed to represent the secondary structure of the hPRL aggregates. Zn2+ causes hPRL aggregation through low-affinity Zn2+-binding sites: hPRL aggregation by addition of Zn2+ has previously been described in outline by solubility (10), sedimentation and DLS studies (12). To describe the


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aggregation process in greater detail, we made use of the difference in secondary structure between the aggregated and the native hPRL (Fig. 2B). hPRL aggregation was followed by measuring θ210 nm immediately after addition of increasing concentrations of Zn2+. Representative aggregation curve demonstrate the quality of the data and the fits (Fig. 3A). Aggregation is largely completed after approx. 30 min. The shape of the curves in Fig. 3A changes around 23 µM Zn2+ from single exponential to double exponential (as shown by the distribution of the residuals, cfr. Fig. S3). The double exponential decay seen > 23 µM Zn2+ suggests an initial fast step followed by a slower step. k1 and k2 plotted against the Zn2+ concentration clearly show that aggregation rate increases dramatically when Zn2+ concentration reaches ~ 25 µM, well above the hPRL concentration of 8.7 µM (Fig. 3B). By the time we reach 25 µM Zn2+, the single high-affinity Zn2+-binding site will be occupied, so that any additional effects (such as the single- and double-exponential decays in Fig. 3) must reflect occupancy of low-affinity Zn2+ binding sites). Thus low-affinity binding sites must be responsible for hPRL aggregation, supporting previous studies by Lee and co-workers where disrupting the hPRL high-affinity Zn2+ binding site did not affect hPRL aggregation (12). Heparin has disaggregase activity due to its ability to bind Zn2+: For in vitro hPRL aggregation to be biologically relevant it has to be reversible. Based on data in Fig. 2C, we investigated heparin as a candidate for hPRL disaggregation at pH 7.4. Aggregates were formed at two different Zn2+ concentrations and the CD ellipticity at 210 nm was followed immediately after addition of increasing heparin concentrations. This gave rise to single exponential decays with rate constants, k, which increased linearly with heparindisaccharide concentration (Fig. 4A). Because the rate of disaggregation increases with heparin concentration, we propose that heparin actively remodels the hPRL aggregates in line with Shan and co-workers (26). We hypothesized that heparin disaggregates hPRL by binding Zn2+. Indeed, EDTA, which binds strongly to Zn2+ (27), is also able to re-solubilize preformed hPRL into its native secondary structure (Fig. S4B) and disaggregation is largely completed after approx. 20 min (Fig. S4A). On a molar basis, EDTA was 100-fold more effective than heparin in disaggregating pre-formed hPRL aggregates (Fig. S4C). We confirmed with ITC that Zn2+ binds to heparin with low affinity. The raw ITC data shows the endothermic binding of Zn2+ to heparin; the upward peaks indicate an uptake of heat from the surroundings (Fig. 4B). The accumulated ∆H was plotted against the [Zn2+]/[heparindisaccharide] molar ratio (lower x-axis) and the total Zn2+ concentration (upper x-axis) for all three pH-values (Fig. 4C). The KD was calculated to ~ 1.2 mM and the interaction approaches completion above Zn2+:heparindisaccharide molar ratios of ~ 4. Similar results have been reported based on potentiometric titrations in the presence of 0.15 M NaCl (28). Together these results suggest


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that the removal of Zn2+ alone causes hPRL to refold and that aggregates of hPRL may dissolve in vivo simply by entering the blood where the concentration of Zn2+ is as low as 20 nM (29). The combination of Zn2+ and GAGs causes hPRL aggregation at pH 6.0 and pH 5.5: hPRL aggregation was also investigated under more acidic conditions, mimicking the conditions in the TGN and in the secretory granules (7). hPRL maintains its native secondary structure at pH 6.0, neither affected by heparin nor Zn2+ when added separately (Fig. S5). The effect of adding heparin and Zn2+ in combination was investigated both at pH 6.0 and 5.5 by keeping the Zn2+ concentration constant, adding increasing concentrations of heparindisaccharide and following aggregation with ellipticity at 210 nm over time until it levelled out. To evaluate the aggregation kinetics, two measures have been used: one is the rate constant, k, derived from the single and double exponential fits, and the other is the end level ellipticity in the cases where no meaningful fits of exponential decays to kinetic data could be obtained. The end level ellipticity (Fig. 5A) value reaches an optimum around equimolar Zn2+:heparin mole ratios (slightly higher at pH 5.5 than at pH 6.0) before it declines to values similar to non-aggregated hPRL. We interpret this to mean that low amounts of heparin neutralize the positive charges of hPRL and thereby enable Zn2+ to bind to and aggregate the protein which gets increasingly positive with decreasing pH. Increasing heparin concentrations eventually halts aggregation, most likely due to heparin’s Zn2+ binding abilities. At constant heparindisaccharide concentration, increasing concentration of Zn2+ caused the aggregation rate constant, k, to increase steadily at pH 5.5 but level off already at ~ 0.3 Zn2+:heparin mole ratios at pH 6.0 (Fig. 5B). hPRL refolds into its native secondary structure by the removal of Zn2+: Having established that both Zn2+ and heparin are needed for hPRL aggregation at pH 6.0 and 5.5, we investigated disaggregation by EDTA at these pH values and compared with the data obtained at pH 7.4 (Fig. 6A). The aggregates were formed with 30 µM Zn2+ at all three pH values. As the pH decreases, the amount of EDTA needed to dissolve the aggregates increases, with the midpoint EDTA:Zn2+ ratio of disaggregation growing from ~0.5 at pH 7.4 to ~1.0 at pH 5.5. Decreasing pH will likely decrease the electrostatic attraction between Zn2+ and hPRL (pI 6.2) but also protonates EDTA (one of whose carboxylates has a pKa of 6.2) and thus weakens interactions between EDTA and Zn2+. Similar aggregated structures are seen across the pH 4.0-7.4 range: TEM images of hPRL aggregates formed at pH 4.0, 5.0, 5.5, 6.0 and 7.4 show similar amorphous aggregates with grainy structure (Fig. 7A), devoid of classical fibrillary morphology. Neither hPRL monomers nor aggregates showed any signs of binding the amyloidspecific dye ThT (Fig. 7B) when compared to ThT-positive fibrils of 0.01 mg/mL α-synuclein. The aggregates gave


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rise to a large trailing peak, indicating a high level of light scattering. CD far-UV spectra (Fig. 8A) and FTIR analysis (Fig. 8B) revealed that the secondary structure of the aggregates was very similar across the pH 4.0-7.4 range, independent of the aggregation-causing agent(s). According to the FTIR spectra, the aggregates still have considerable α-helix structure with minima at 1548 cm-1 and 1653 cm-1 (Fig. 8B) (30). hPRL stability reaches a minimum around pH 5.5: Over the pH range 4-9, hPRL thermal stability shows a clear minimum around pH 5.5, where the Tm is decreased by around 12˚C compared to the optimal stability at pH 7.4 (Fig. 9). Adding GAGs to hPRL strongly destabilizes the protein below ~ pH 6.5, and the Tm at pH 5.5 declines to

53.4±0.1˚C, which is 14˚C lower than that of GAG-free hPRL at the same pH. Below pH 5.5, hPRL aggregates in the presence of GAGs and it is not possible to determine any thermal transition. Discussion Proposed mechanism of hPRL aggregation, storage and re-solubilization: Based on our results, we propose the following mechanism for hPRL’s aggregation, storage and re-solubilization during its passage through the secretory pathway (summarized in Fig. 10). After ribosomal synthesis, hPRL is directed to the ER in a soluble state. In this neutral compartment (pH ~ 7.4) no aggregates are expected to form as no significant self-association of hPRL is seen at concentrations up to 250 µM (10). However, should hPRL encounter Zn2+ at > 2-fold molar excess at this neutral pH, hPRL will change conformation (Fig. 2) and aggregate through its low-affinity Zn2+ binding sites (Fig. 3, (12)). This, however, is unlikely because no free zinc is available for this low-affinity aggregation in the ER of protein secreting cells of the rat anterior pituitary (11). From the ER, hPRL moves through the Golgi complex to the TGN where the pH is lowered to 6.0 (7) and the concentration of hPRL likely increases as observed for rat PRL (8). This acidification could be expected to affect the stability of hPRL and does in fact destabilize the protein by approx. 10˚C (Fig. 9). The change from pH 7.4 to pH 6.0 also changes the overall charge of hPRL from negative (-4.2) to slightly positive (+1.5) as calculated with the InCharge software (Aptum Biologics Ltd., UK). In the TGN, hPRL encounters free Zn2+ (in detectable levels (11)) as well as GAGs (31, 32). The combination of the two results in rapid hPRL aggregation (Fig. 5). Aggregation is largely completed after 30 minutes (Fig. 3A), which nicely agrees with in vivo observations where hPRL aggregates are observed in the Golgi complex ~ 30 minutes after pulse labelling pituitary cells with [3H]leucine (8). The hPRL aggregates are ThT-negative and have an amorphous appearance as seen by TEM (Fig. 7). The secondary structure of the aggregates is similar across the pH 7.4-4.0 range, independently of the aggregation-causing agent(s) (Fig. 8). This common structure might enable the TGN membrane proteins to 12 ACS Paragon Plus Environment

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Biochemistry

recognize hPRL aggregates, leading to the initial sorting of PRL aggregates within the Golgi cisternae. Destabilization of hPRL by GAGs is an indication that GAGs preferentially bind to non-native (e.g. thermally or acid-denatured) states of hPRL. The proteins are not in random coil structure according to CD and FTIR, but probably form a relatively extended structure, which allows hPRL to associate via hydrophobic interactions (Fig. 2A) and provides access for GAG binding to hPRL positive charges. As the mature hPRL granules are formed, the pH is lowered to 5.5 (7) which causes the aggregation rate to increase (Fig. 5B) and all available hPRL to aggregate and remain in the granules, thus completing the sequestration of hPRL. In response to an appropriate signal, the secretory granules release their content by exocytosis into the blood where the concentration of Zn2+ is lowered to ~ 20 nM (29) and the pH returns to neutral. These conditions result in solubilization of the granular core, suggested to be caused by the removal of Zn2+, as mimicked by EDTA (Fig. 6A). The disaggregation by EDTA was largely completed after 20 minutes (Fig. S2B) in good correlation with in vitro experiments performed on isolated membrane-less rat PRL granules where half of the protein core is solubilized within 20 min at neutral pH (33). Similarly, the removal of Zn2+ by GAGs enables hPRL to refold into its monomeric, native secondary structure (Fig. 6B), necessary for proper biological function. The structural epitope: The sorting mechanism for hPRL – and other secretory proteins – is still obscure. It has been suggested that aggregates are recognized by the appropriate membrane proteins on the basis of some structural feature (34) since some cells are able to differentiate between aggregates of different protein origin (35). A structural epitope of hPRL – and 32 other peptide hormones – has been suggested to be the cross-β structure of amyloid fibrils formed in vitro (17). However, our results show no evidence of amyloid structures as the aggregates did not bind ThT (Fig. 7B), no amyloid-characteristic peak in the 1615-1630 cm-1 area was seen in the FTIR analysis (Fig. 8B) and no fibrillary structures were identified by TEM (Fig. 7A). We instead saw aggregated structures which formed quickly and retained some degree of secondary structure. We speculate that the striking similarity between the aggregates, irrespective of the aggregation-causing agent(s), entails a structural motif that may be recognized by membrane proteins in the Golgi, causing them to be appropriately packed in secretory granules. Supporting this, a similar secondary structure, with the single 227 nm minimum, has also observed in the case of salmon calcitonin – another hormone stored in secretory granules of parafollicular cells (36, 37). We suggest the pattern of hPRL aggregation presented here to be biologically relevant not only due to the reversibility of the reactions where the protein refolds into its native – and presumable active – conformation, but also because the aggregation and disaggregation processes occur on the same minute-level time scale in vivo and in vitro.


Biochemistry

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The role of GAGs in protein aggregation: GAGs are common in prolactin granules as well as in granules of other secretory proteins (15), indicating a crucial role for GAGs in granule formation. Our results suggest that the GAGs facilitate hPRL aggregation by neutralizing the positive charges on hPRL. As the environment acidifies, as experienced by proteins through the secretory pathway, the number of positive surface charges will increase, causing repulsion between molecules. Because of the potential role of GAGs in secretory granule formation and their ability to induce amyloid fibril formation of proteins normally stored in such granules (17, 38), we speculate that GAGs are involved in the fine-tuning between the formation of storage-relevant aggregates and the formation of pathological amyloids. This has been suggested to be the case for amylin, involved in diabetes type II, where the formation of fibrils is dependent on the backbone and sulfation degree of GAGs (39). Our results also indicate that hPRL:GAG interactions are sensitive to the sulfate content or the structure of the GAG since heparin and CSA interact with hPRL in slightly different ways at neutral pH where heparin (highly sulfated) forms stable complexes with hPRL while CSA (less sulfated) does not (Fig. 1A). In addition, mixing hPRL with Zn2+ before the addition of GAGs at neutral pH results in mixing order hysteresis (incomplete disaggregation) for CSA but not heparin (data not shown). Under other conditions, hPRL is reported to form ThT positive fibrils in the presence of CSA but not heparin (17). Biologically, these hPRL fibrils might be a consequence of aging as amyloids with hPRL origin has been identified in patients with systemic amyloidosis (40). Acknowledgements We are very grateful to Dr. Birthe Kragelund for the gift of the hPRL expression plasmid and for constructive discussions about hPRL in general. Supporting Information Available This consists of five sets of figures which show calibration of the thiol quantification assay and the secondary structure of hPRL at pH 5.0-7.4 (Fig. S1), the effect of heparin on Zn2+-induced changes in hPRL secondary structure (Fig. S2), kinetic profiles of the aggregation of hPRL in the presence of Zn2+ (Fig. S3), kinetic profiles and CD spectra of the disaggregation of hPRL by EDTA (Fig. S4) and the inability of heparin and Zn2+ to alter the secondary structure of hPRL at pH 6.0 (Fig. S5).


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K. Teilum, J. C. H., V. Goffin, S. Kinet, J. A. Martial, B. B. Kragelund. (2005) Solution Structure of Human Prolactin, Journal of Molecular Biology 351, 810-823. M. J. K. Hansen, J. G. O., S. Bernichtein, C. O'Shea, B. W. Sigurskjold, V. Goffin, B. B. Kragelund. (2010) Development of prolactin receptor antagonists with reduced pH-dependence of receptor binding, Journal of Molecular Recognition 24, 533-547. C. N. Pace, F. V., L. Fee, G. Grimsley, T. Gray. (1995) How to measure and predict the molar absorption coefficient of a protein, Protein Science 4, 2411-2423. M. Oliveberg, S. V., A. R. Fersht. (1994) Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state, Biochemistry 33, 8826-8832. I. Lazar, I. L. (2010) Gel Analyzer 2010a: Freeware 1D gel electrophoresis image analysis software, Available online: http://www.gelanalyzer.com. S. Kinet, V. G., V. Mainfroid, J. A. Martial. (1996) Characterization of Lactogen Receptor-binding Site 1 of Human Prolactin, The Journal of Biological Chemistry 271, 14353-14360. J. L. Voorhees, G. V. R., T. J. Gordon, C. L. Brooks. (2011) Zinc binding to human lactogenic hormones and the human prolactin receptor, FEBS Letters 585, 1783-1788. P. Jaru-Ampornpan, K. S., V. Q. Lam, M. Ali, S. Doniach, T. Z. Jia, S. Shan. (2010) ATP-independent reversal of a membrane protein aggregate by chloroplast SRP subunit, Nature Structural & Molecular Biology 17, 696-703. M. Kandeel, T. Y., M. Al-Julaifi, A. AL-Rizki, Y. Kitade. (2013) Chelating efficiency and mechanisms of interaction of some toxic and biologically important cations with EDTA by isothermal titration caliometry, Life Science Journal 10, 2042-2047. D. Grant, W. F. L., F. B. Williamson. (1992) Zn2+-heparin interaction studied by potentiometric titration, Biochemical Journal 287, 849-853. C. J. Frederickson, L. J. G., A. Krezel, D. J. McAdoo, R. N. Muelle, Y. Zeng, R. V. Balaji, R. Masalha, R. B. Thompson, C. A. Fierke, J. M. Sarvey, M. de Valdenebro, D. S. Prough, M. H. Zornow. (2006) Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion, Experimental Neurology 198, 285-293. D. M. Byler, H. S. (1986) Examination of the secondary structure of proteins by deconvoluted FTIR spectra, Biopolymers 25, 469-487. J. E. Silbert, G. S. (2002) Biosynthesis of chondroitin/dermatan sulfate, IUBMB Life 54, 177-186. M. Perez, C. B. H. (1986) Transport of sugar nucleotides and adenosine 3'-phosphate 5'-phosposulfate into vesicles derived from the Golgi apparatus, Biochimica et Biophysica Acta 864, 213-222. G. Giannattasio, A. Z., J. Meldolesi. (1975) Molecular organisation of rat prolactin granules. I. In vitro stability of intact and "membraneless" granules, The Journal of Cell Biology 64, 246-251. C. Keeler, M. E. H., P. S. Dannies. (2004) Is there structural specificity in the reversible protein aggregates that are stored in secretory granules?, Journal of Molecular Neuroscience 22, 43-49. M. S. Lee, Y. L. Z., J. E. Chang, P. S. Dannies. (2001) Acquisition of Lubrol Insolubility, a Common Step for Growth Hormone and Prolactin in the Secretory Pathway of Neuroendocrine Cells, The Journal of Biological Chemistry 276, 715-721. G. Siligardi, B. S., S. Melandri, M. Visconti, A. F. Drake. (1994) Correlations between biological activities and conformational properties for human, salmon, eel, porcine calcitonins and Elcatonin elucidated by CD spectroscopy, European Journal of Biochemistry 221, 1117-1125. Diociaiuti, M., Gaudiano, M. C., and Malchiodi-Albedi, F. (2011) The slowly aggregating salmon Calcitonin: a useful tool for the study of the amyloid oligomers structure and activity, Int J Mol Sci 12, 9277-9295.


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S. Jha, S. M. P., J. Gibson, C. E. Nelson, N. N. Adler, A. T. Alexandrescu. (2011) Mechanism of amylin fibrillization enhancement by heparin, The Journal of Biological Chemistry 286, 22894-22904. G. M. Castillo, J. A. C., W. Yang, M. E. Judge, M. J. Sheardown, K. Rimvall, J. B. Hansen, A. D. Snow. (1998) Sulfate content and specific glycosaminoglycan backbone of perlacan are critical for perlacan's enhancement of islet amyloid polypeptide (amylin) fibril formation, Diabetes 47, 612-620. P. Westermark, L. E., U. Engström, S. Eneström, K. Sletten. (1997) Prolactin-derived amyloid in the aging pituitary gland, Americal Journal of Pathology 150, 67-73.

Figure legends Figure 1 Complex formation between hPRL and heparin – but not CSA – at pH 7.0 does not affect hPRL stability. A, B: SEC analysis of hPRL at pH 7.0 (A) or pH 7.4 (B) either alone or with GAGs. hPRL was incubated alone or in the presence of heparin or CSA in 1:35 and 1:45 molar ratios, respectively, prior to analysis. Only a low degree of complex formation was seen between hPRL and heparin when increasing the pH to 7.4. C: CD thermal denaturation of hPRL with or without GAGs. Heparin binding does not affect the thermal stability of hPRL. The ratios are in molar concentrations. Figure 2 Zn2+-induced hPRL conformational changes are counteracted by GAGs at pH 7.4. A: ANS analysis of monomeric (black) and aggregated hPRL (green). Pre-incubating of 8.7 µM hPRL at pH 7.4 with increasing concentrations of CSA suppresses the Zn2+-induced conformational changes of hPRL. A model with two consecutive binding steps (hPRL + CSA hPRL:CSA; hRPL:CSA+CSA hPRL:CSA2 with associated dissociation constants Kd1 and Kd2) was fitted to the data, yielding an apparent Kd1 value of 7.8 ± 1.1 µM. Kd2 could not be determined with sufficient accuracy due to the slow decline over the probed concentration range. In the shown fit, it is fixed at 300 µM but this is merely for illustrative purposes. B: CD far-UV spectra showing the protective role of CSA on Zn2+-induced change in hPRL secondary structure. C: hPRL was pre-incubated with increasing concentrations of GAGs before aggregation with Zn2+ and SDS-PAGE of the pellet and supernatant fraction. Quantification shows the ability of GAGs to keep hPRL solubilized. Figure 3 hPRL aggregates through low-affinity Zn2+-binding sites at pH 7.4. A: Aggregation of hPRL with increasing Zn2+ concentrations was followed over time with CD. Curves were fitted to either single or double exponentials to determine rate constants of aggregation. B: The effect on aggregation rate constants, estimated from the curves in 17 ACS Paragon Plus Environment

Biochemistry

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(A), with increasing Zn2+ concentrations. Initiation of hPRL aggregation requires more than 2-fold molar excess of Zn2+. Figure 4 Heparin has disaggregase activity due to its ability to bind Zn2+. A: The ability of heparin to dissolve preformed hPRL aggregates at pH 7.4 was shown with CD. Aggregates were formed with hPRL and Zn2+ in 1:3.5 or 1:4.5 molar ratios prior to addition of heparin. B, C: A plot of the raw data at pH 6.0 (B) and of the accumulated ∆H at pH 7.4, 6.0 and 5.5 (C) from ITC analysis of the binding between heparin and Zn2+. The KD of binding (= 1.2 mM) was calculated from (C). Figure 5 The combination of Zn2+ and GAGs causes hPRL aggregation at pH 6.0 and pH 5.5. A, B: Aggregation of hPRL at pH 6.0 (black) and 5.5 (blue) was followed over time with CD by pre-incubating hPRL with Zn2+ and adding increasing heparin concentrations (A) or by pre-incubating hPRL with heparin and adding increasing Zn2+ concentrations (B). This is reflected in the units of the two x-axes. The end level for (A) was visually estimated after reaction completion. Rate constants in (B) were estimated from single exponential fitting. A slightly higher GAG concentration is needed for optimal aggregation of hPRL at pH 5.5 compared to pH 6.0. Figure 6 hPRL refolds into its native secondary structure by the removal of Zn2+. A: Disaggregation was performed with EDTA on pre-formed hPRL aggregates at pH 7.4, 6.0 and 5.5 and followed by CD over time. The end level value was estimated after reaction completion. B: The ability of hPRL to refold into its native secondary structure by the removal of Zn2+ was shown with far-UV CD. Figure 7 hPRL aggregates are amorphous and ThT-negative. A: TEM analysis of hPRL aggregates formed at pH 7.4, 6.0, 5.5, 5.0 and 4.0. Similar morphologies are seen. B: ThT analysis of hPRL monomers and hPRL aggregates at pH 7.4, 6.0 and 5.5 show that neither the monomer nor the aggregates bind ThT. Figure 8


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Biochemistry

hPRL aggregates are similar in structure across the pH 4.0-7.4 range. A: Far-UV CD spectra show the similarity in secondary structure of hPRL aggregates formed at different pH-values. B: Similar structures of hPRL aggregates are seen with FTIR analysis, independent of pH and aggregation-causing agent(s). The spectra reveal considerable α-helical secondary structure. Figure 9 hPRL stability reaches a minimum around pH 5.5. A: Melting temperatures of hPRL either alone (black) or in 1:35 or 1:45 molar ratios with heparin (blue) and CSA (green), respectively, across the pH 4-9 range. Native hPRL has a stability minimum at pH 5.5, while GAGs have an increasingly destabilizing effect on hPRL with decreasing pH, leading to aggregation below pH 5.5. Figure 10 Proposed mechanism of hPRL aggregation, storage and re-solubilization. hPRL in its aggregated conformation.


, natively folded hPRL.

,

Biochemistry

Figures Figure 1

A

hPRL:heparin interactions hPRL alone, pH 7.4 hPRL + hep, pH 7.4

280 nm

(mAU)

25

15

Absorbance

(mAU)

30

hPRL alone, pH 7.0 hPRL + hep, pH 7.0 hPRL + CSA, pH 7.0 CSA alone, pH 7.0

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Figure 2

A

B Changes in hPRL secondary structure

ANS fluorescence

0

and CSA

hPRL alone

-5

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140 120 100

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160

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Protection against aggregation CSA Heparin y = 0.92167 - 0.25161x R= 0.96676 y = 0.88894 - 0.3466x R= 0.97658

0.8

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1.6

2

] (mM)


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Figure 3

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hPRL aggregation with increasing Zn

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Figure 4

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[Zn ]/[heparin

disaccharide

1.2

1.4

]

Figure 6

A

B

Disaggregation with EDTA

0

-10

-5

-1

-15

Secondary structure after refolding pH 7.4 pH 6.0 pH 5.5

-1

pH 7.4 pH 6.0 pH 5.5


-5

-20

-10

2

Plateau (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

-25 -30 -35 -40 0

0.2

0.4

0.6

0.8

1 2+

EDTA/[Zn ]

1.2

1.4

1.6

-15

-20

-25 200

210

220

230

Wavelength (nm)


240

250

Page 25 of 28

Figure 7

A pH 4.0

pH 5.0

pH 6.0

pH 7.4

B hPRL aggregates are ThT negative Monomer, pH 7.4 Monomer, pH 6.0 Monomer, pH 5.5 Aggregates, pH 7.4 Aggregates, pH 6.0 Aggregates, pH 5.5 0.01 mg/mL alpha-synuclein fibrils

70

ThT fluorescence emission (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

60 50 40 30 20 10 0 460

480

500

520

540

Wavelength (nm)


pH 5.5

Biochemistry

Figure 8

A

B

Aggregated secondary structure

Aggregated secondary structure

1

-1


5

0

-1

Absorbance (a.u.)

0

2

-5

-10

-1

-2

-3 pH 4.0

pH 4.0 pH 5.0 pH 5.5 pH 6.0 pH 7.4

-15

-20

210

220

230

240

pH 5.5

-4

pH 6.0

250

-5 1520

pH 7.4

1560

1600

Figure 9

85

Effect of GAGs + pH on hPRL stability

m

80 75 70 65 60 55 50

hPRL alone hPRL + heparin hPRL + CSA

4

5

6

1640

Wavenumber (cm-1)

Wavelength (nm)

Melting temperature, T (C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

7

8

9

pH


1680

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 10

Cell membrane

TGN

Secretory granule


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

For Table of Contents Use Only

A complex dance: the importance of glycosaminoglycans and zinc in the aggregation of human prolactin Line Friis Bakmann Christensen1, Kirsten Gade Malmos1, Gunna Christiansen2 and Daniel Erik Otzen*1

Cell membrane

TGN

Secretory granule


A Complex Dance: The Importance of ... - ACS Publications

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