Báo cáo khoa học: Casein phosphopeptide promotion of calcium uptake in HT-29 cells ) relationship between biological activity and supramolecular structure ppt

Casein phosphopeptide promotion of calcium uptake in HT-29 cells ) relationship between biological activity and supramolecular structure Claudia Gravaghi1, Elena Del Favero1, Laura Cantu’1, Elena Donetti2, Marzia Bedoni2, Amelia Fiorilli1, Guido Tettamanti1 and Anita Ferraretto1 Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy DMU, Department of Human Morphology, University of Milan, Italy Keywords Ca2+ uptake; casein phosphopeptides; casein phosphopeptide–Ca2+ aggregates; HT-29 cells; laser light scattering Correspondence A Ferraretto, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, L.I.T.A via F Cervi 93, 20090 Segrate, Italy Fax: +39 02 50330365 Tel: +39 02 50330374 E-mail: anita.ferraretto@unimi.it (Received 22 May 2007, revised July 2007, accepted 27 July 2007) doi:10.1111/j.1742-4658.2007.06015.x Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT-29 cells that have been shown to be differentiated in culture The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs b-CN(1–25)4P and as1-CN(59–79)5P, the commercial mixture CPP DMV, the ‘cluster sequence’ pentapeptide, typical of CPPs, and dephosphorylated b-CN(1–25)4P, [b-CN(1–25)0P] The biological effect was found to be: (a) maximal with b-CN(1–25)4P and null with the ‘cluster sequence’; (b) independent of the presence of inorganic phosphate; and (c) maximal at mmolỈL)1 Ca2+ The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by b-CN(1–25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+-free Krebs ⁄ Ringer ⁄ Hepes; (e) formation of bigger aggregates (150 nm radius) with b-CN(1–25)0P With both b-CN(1–25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at mmolỈL)1 Ca2+ It is known that milk is an excellent source of bioavailable calcium, due to the presence of caseins, which bind calcium, keeping it in a soluble and absorbable state [1–5] In bovine milk, about two-thirds of the calcium and one-half of the inorganic phosphate are bound to various species of caseins, aS1-casein, aS2-casein, b-casein, and k-casein, forming colloidal micelles with a calcium ⁄ phosphate ⁄ casein molar ratio of 30 : 21 : [6] The casein micelles, of about 100 nm radius, are stable structures composed of hundreds of smaller aggregates, named calcium phosphate nanoclusters, or nanocomplexes, having a core of calcium phosphate surrounded by a shell of casein molecules [7–10] The portion of the casein molecule responsible for the ability to maintain calcium and phosphate ions in a soluble form are amino acid sequences containing the common motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu (the ‘cluster sequence’ or ‘acidic motif’) Peptides containing this sequence (casein phosphopeptides, CPPs) are produced in vivo from the digestion of aS1-casein, aS2-casein and b-casein by gastrointestinal proteases [11–13], and in vitro by tryptic and chimotryptic fragmentation of casein followed by precipitation [14] Calcium phosphate nanoclusters (or complexes) were also prepared and physicochemically characterized using CPPs, namely b-CN(1–25)4P and b-CN(1– 42)5P, corresponding to the first 25 or 42 amino acids of b-casein, respectively, and aS1-CN(59–79)5P, Abbreviations ALP, alkaline phosphatase; BrdU, bromodeoxyuridine; [Ca2+]i, intracellular free calcium concentration; [Ca2+]o, extracellular free calcium concentration; CN, casein; CPP, casein phosphopeptide; CPP DMV, CPP of commercial origin; KRH, Krebs ⁄ Ringer ⁄ Hepes FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS 4999 CPP activity and supramolecular structure C Gravaghi et al corresponding to the sequence 59–79 of aS1-casein [8,9,14–16] A few years ago, we showed that a CPP mixture of commercial origin with five main components, as well as pure b-CN(1–25)4P and even, to a lesser extent, aS1-CN(59–79)5P elicited a marked and transient rise of intracellular free Ca2+ concentration ([Ca2+]i) in human intestinal tumor HT-29 cells differentiated in culture [17] The intracellular Ca2+ rise caused by CPP was due to uptake of extracellular calcium ions, with no involvement of the intracellular calcium stores [17] A subsequent study, performed with b-CN(1–25)4P and some chemically synthesized peptides corresponding to precise fragments of the b-CN(1–25)4P sequence, clarified that a well-defined primary structure is required for the bioactive response [18] This structure includes the N-terminal portion characterized by the presence of a loop and a b-turn, and the ‘cluster sequence’ However, and notably, the ‘cluster sequence’ alone does not exhibit the Ca2+ uptake effect, suggesting that a particular supramolecular structure of CPP–Ca2+ complexes is required for the observed biological effect in vitro, by analogy with the relationship between calcium phosphate–CPP aggregation as nanoclusters and the capacity to bind and maintain calcium in a bioavailable form The present investigation addressed the question whether a supramolecular structure of CPP–Ca2+ is needed to stimulate Ca2+ uptake by differentiated HT-29 cells To this end, we first tested whether, under the conditions used to prepare calcium phosphate–CPP nanoclusters [16], the [Ca2+]i-increasing effects of CPP on HT-29 cells could be detected Unfortunately, these conditions were not suitable for the growth of HT-29 cells in culture Therefore, we adopted the same experimental conditions previously used to detect the biological effects of CPPs, that is: (a) the individual CPPs b-CN(1–25)4P and as1-CN(59–79)5P, and the commercial mixture CPP DMV; (b) HT-29 human colon carcinoma cells, differentiated in culture; (c) a Krebs ⁄ Ringer ⁄ Hepes (KRH) solution buffering the cells at pH 7.4, containing given concentrations of Ca2+ (as CaCl2), with or without phosphate (as KH2PO4), compatible with normal cell viability; and (d) CPP concentrations that have been shown to affect Ca2+ uptake by the cells [17,18] The possible occurrence under these conditions of a supramolecular structural organization (aggregation) of CPP and Ca2+ was studied by a laser light scattering technique capable of establishing the dimensions (hydrodynamic radius) and the relative amounts of aggregates in solution Care in exactly matching the experimental conditions for laser light scattering experiments with 5000 those providing the mentioned biological effect of CPP was of central importance Results In our previous work [17], we demonstrated that CPPs are able to promote Ca2+ uptake by human intestinal HT-29 tumor cells differentiated in culture (RPMI) with a consequent transient rise of [Ca2+]i In order to address the question whether a supramolecular structural organization of CPP–Ca2+ is needed to promote this biological effect, we first verified the differentiation state of HT-29 cells in culture It is known that HT-29 cells cultured in DMEM with a high d-glucose content (25 mmolỈL)1) not present signs of spontaneous differentiation towards intestinal-like cells [19] Instead, when the culture medium is switched to RPMI, with low d-glucose concentration (13.9 mmolỈL)1), or to a DMEM medium with galactose gradually substituting for glucose, HT-29 cells undergo a process of intestinal-like differentiation [20] On this basis, HT-29 cells were cultured in RPMI (low d-glucose) or galactosecontaining medium, and their differentiation was assessed by determining specific biochemical markers [alkaline phosphatase (ALP) and sucrase-isomaltase] and the rate of proliferation, and by electron-microscopic examination As shown in Fig 1A, the levels of ALP and sucrase-isomaltase in RPMI cells were not significantly different from those in DMEM cells (631 ± 32 versus 623 ± 25 mmg)1 protein for ALP, and 80.7 ± 9.1 versus 77.8 ± 8.3 mmg)1 protein for sucrase-isomaltase, respectively), whereas galactose-adapted cells showed a marked increase of both ALP (830 ± 12 mmg)1 protein) and sucraseisomaltase (270 ± 20 mmg)1 protein) The proliferation rate (Fig 1B) of cells cultured in RPMI and galactose-adapted medium markedly decreased as compared to DMEM cells, indicating a repression of their tumoral condition The cell morphology is shown in Fig 1C–E DMEM cells appear to be completely devoid of apical microvilli and junctional apparatus, whereas RPMI cells present a well-developed brush border, with microvilli on their apical side, together with the presence of adherent junctions and desmosomes, and galactose-adapted cells display all the features observed in RPMI cells, with, in addition, characteristic intracellular laminae surrounded by numerous and well-developed small microvilli All of these findings indicate that HT-29 cells grown in RPMI or galactose-containing medium undergo a remarkable process of intestinal-like differentiation, confirming previous data [19,20] From both the quantitative and qualitative points of view, both FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gravaghi et al CPP activity and supramolecular structure mU/mg protein A ALP sucrase 1000 * 750 500 * 250 DMEM RPMI GALACTOSE Fig HT-29 cell differentiation (A) ALP (white bars) and sucrase (black bars) enzyme activities of DMEM (undifferentiated), RPMI and galactose-adapted (differentiated) cells (B) Proliferation rate as determined by BrdU incorporation in the three cell populations expressed as percentage with respect to DMEM cells (C,D,E) Transmission electronmicrographs of araldite ultrathin sections of DMEM cells (C), RPMI cells (D) and galactose-adapted cells (E), respectively Starting from the apical side, arrows in (D) indicate adherent junctions and desmosomes Original magnification: (C,D) ·10 000; (E) ·14 000 Data reported in (A) and (B) represent mean value ± SD (n ¼ 5–6 experiments for each bar) Asterisks indicate significantly different values (P < 0.05) from DMEM BrdU incorporation (%) B 100 * * 50 DMEM C RPMI and galactose-adapted cells responded equally to CPP administration, with an increase of [Ca2+]i Notably, undifferentiated HT-29 cells did not exhibit the CPP effect (unpublished results) On this basis and for purposes of simplicity, all further experiments were performed by culturing cells in RPMI medium To investigate the effect of CPP in increasing the extracellular free Ca2+ concentration ([Ca2+]o) in the buffer solution, while avoiding the possible precipitation of insoluble calcium phosphate salts, which would affect biological and laser light scattering measurements, we first explored whether the presence of phosphate was necessary for the biological effect of CPP To this end, a first dose–response set of experiments at [Ca2+]o higher than mmolỈL)1 was performed using cells grown in RPMI As shown in Fig 2A,B the [Ca2+]i peaks of increase in HT-29 cells elicited by b-CN(1–25)4P CPP at two different concentrations (50 and 100 lmolỈL)1) and in the presence of or mmolỈL)1 [Ca2+]o, expressed as percentage of the basal RPMI D GALACTOSE E values, were the same regardless of the presence or absence of phosphate In more detail (Fig 2C,D), for mmolỈL)1 [Ca2+]o and 50 lmolỈL)1 b-CN(1–25)4P, the basal Ca2+ concentration was 100 nmolỈL)1 in the presence of phosphate (trace a) and 70 nmolỈL)1 in the absence of phosphate (trace b), whereas the increments due to CPP were 25 nmolỈL)1 and 22 nmolỈL)1, respectively; for mmolỈL)1 [Ca2+]o and 100 lmolỈL)1 b-CN(1–25)4P, the basal Ca2+ concentration was 72 nmolỈL)1 (trace c) and 84 nmolỈL)1 (trace d), whereas the increments due to CPP were 48 nmolỈL)1 and 47 nmolỈL)1, respectively, i.e the same regardless of the presence or absence of phosphate in the buffer Similar results were obtained with the CPP DMV mixture and as1-CN(59–79)5P, indicating that free phosphate is not involved in the biological effect of CPP More details on the dose–response relationship (in the absence of phosphate) are presented in Fig 3, where [Ca2+]o was raised to mmolỈL)1 and the three different preparations of CPP, each at different concentrations, were employed With b-CN(1–25)4P and FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS 5001 CPP activity and supramolecular structure C Gravaghi et al KRH (containing phosphate) phosphate-free KRH A B 100 [Ca2+]o 2mmol/L 100 [Ca2+]o 4mmol/L peak calcium increase (% on basal value) * * 50 50 0 50 100 50 100 [β-CN(1-25)4P] μmol/L [β-CN(1-25)4P] μmol/L C D 600 Fluorescence arbitrary units * * a b [β-CN(1-25)4P] 50 μmol/L 600 c d [β-CN(1-25)4P] 100 μmol/L Ionomycin 400 Ionomycin 400 200 200 100 200 100 200 Time (s) Fig Intracellullar Ca2+ increases in response to administration of b-CN(1–25)4P peptide in KRH or in phosphate-free KRH The data were collected on fura-2-loaded HT-29 cell populations grown in RPMI and treated with two CPP concentrations (50 and 100 lmolỈL)1) and at two different extracellular Ca2+ concentrations, mmolỈL)1 (A) and mmolỈL)1 (B) HT-29 cells were resuspended, just before the experiment, in KRH (black bars) or phosphate-free KRH (white bars) The data collected were expressed as the mean value of [Ca2+]i peak rise (calculated as percentage on basal value) ± SD (n ¼ 3–4 experiments for each bar) Asterisks indicate significantly different values (P < 0.05) from the minimal CPP dose In (C) and (D), the representative traces relative to 50 lmolỈL)1 b-CN(1–25)4P (arrow) in KRH (trace a), and in phosphatefree KRH (trace b), and the representative traces relative to 100 lmolỈL)1 b-CN(1–25)4P (arrow) in KRH (trace c), in phosphate-free KRH (trace d) at mmolỈL)1 extracellular Ca2+ concentration, are shown The vertical scale indicates fluorescent intensity at 485 nm emission wavelength after excitation at 343 nm CPP DMV mixture, the highest biological effects were observed at mmolỈL)1 [Ca2+]o (Fig 3A,B), the optimal effect being obtained at 200 lmolỈL)1 b-CN(1– 25)4P and 1280 lmolỈL)1 CPP DMV, respectively The differences between CPP DMV and b-CN(1–25)4P doses may be explained by considering that, whereas b-CN(1–25)4P is a synthetic, pure peptide, CPP DMV is a mixture of peptides with different primary sequences, and possibly different biological efficacies The behavior of as1-CN(59–79)5P, reported in Fig 3C, appears to be completely different First of all, the extent of the measured effect is much more limited, over the whole CPP and [Ca2+]o concentration range explored Second, the highest activity, within the inves5002 tigated range of Ca2+ concentration (2–6 mmolỈL)1), was recorded at mmolỈL)1 [Ca2+]o Third, no significant change in [Ca2+]i was observed when the CPP concentration was increased Notably, the absence of phosphate in the culture media did not modify cell morphology and viability Also surprising was the finding that when CPP was added to the cell-containing mixtures before the addition of Ca2+, no [Ca2+]i rise was recorded in HT-29 cells due to the presence of CPP It should be remembered that the ‘cluster sequence’ is completely unable to elicit the increase in [Ca2+]i [18] Preliminary laser light scattering experiments showed that an aqueous solution of b-CN(1–25)4P, as well as FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gravaghi et al CPP activity and supramolecular structure A 300 [β-CN(1-25)4P] 50 200 μmol/L 150 μmol/L 100 μmol/L 50 μmol/L I-1 r 150 25 peak [Ca2+]i increase (% on basal value) B [CPP DMV] 300 1280 μmol/L 960 μmol/L 640 μmol/L 320 μmol/L 150 C 300 [αs1-CN(59-79)5P] 200 μmol/L 150 μmol/L 100 μmol/L 50 μmol/L 150 [Ca2+]ommol/L Fig CPP bioactivity is related to extracellular Ca2+ and peptide concentration The data were collected after administering to fura2-loaded HT-29 cell populations various amounts of individual CPPs, b-CN(1–25)4P and as1-CN(59–79)5P, (A) and (C), respectively, and of a mixture of CPPs (CPP DMV) (B) Each point on the graphs corresponds to the mean value of the [Ca2+]i peak rise ± SD, obtained from three or four experiments, and expressed as a percentage of the basal value; in all cases, a CPP single dose was provided to the cells at a fixed extracellular Ca2+ concentration All values are significantly different from each other (P < 0.05) of CPP DMV, as1-CN(59–79)5P, b-CN(1–25)0P and the ‘cluster sequence’, at the used concentrations, gave a very low scattered intensity, similar to that of pure solvent, indicating a condition where aggregation is absent Therefore, the CPP solution in water can be considered a full monomer solution of CPP In contrast, the solution of the same CPP in phosphate-free or phosphate-containing KRH with no Ca2+ showed a remarkable increase of scattered light, of the order of 10 times that of the pure solvent, indicating the occurrence of some aggregation An additional fourfold increase of the scattered light occurred when the solvent contained mmolỈL)1 [Ca2+]o, whereas addition of Ca2+ to a pre-existing Ca2+-free CPP solution did not induce any increase of scattered light (data are shown in Fig 4) The time needed for the occurrence β-CN(1-25)4P β β-CN(1-25)0P Fig Excess scattered intensity relative to the solvent, Ir ) 1, for: 1, b-CN(1–25)4P in phosphate-free KRH containing mmolỈL)1 Ca2+ 2, b-CN(1–25)4P in phosphate-free KRH without Ca2+; 3, b-CN(1–25)4P prepared in phosphate-free KRH without Ca2+ followed by addition of mmolỈL)1 Ca2+; 4, b-CN(1–25)0P in phosphate-free KRH containing mmolỈL)1 Ca2+; 5, b-CN(1–25)0P in phosphate-free KRH without Ca2+ (for all solvents, phosphate-free KRH with or without mmolỈL)1 Ca2+, the same very small scattered intensity was measured) of aggregation corresponded to the duration of the experimental manipulations (pipetting, mixing, etc.), i.e a few seconds This indicates that the process of aggregation, when it occurs, is very rapid b-CN(1– 25)0P, the dephosphorylated form of b-CN(1–25)4P, dissolved in phosphate-free KRH gave rise to a higher scattered intensity with respect to the corresponding b-CN(1–25)4P solution, but no significant influence of Ca2+ was observed (Fig 4), suggesting the occurrence of an aggregation process different from that of b-CN(1–25)4P Finally, the ‘cluster sequence’ did not exhibit any aggregation in solution, regardless of the presence of Ca2+, as its scattered intensity was not dissimilar to that of pure solvent Dynamic light scattering experiments showed (Table 1) that the three CPPs, b-CN(1–25)4P, as1CN(59–79)5P and CPP DMV, dissolved in mmolỈL)1 Ca2+ phosphate-free KRH, formed aggregated structures with the same hydrodynamic radius (RH ¼ 60 ± nm) An identical hydrodynamic radius was detected for the aggregates of b-CN(1–25)4P dissolved in phosphate-free KRH in the the absence of Ca2+ Instead, b-CN(1–25)0P formed much bigger aggregates (RH ¼ 150 ± nm), regardless of the presence or absence of Ca2+ Concerning the three CPPs with the same hydrodynamic radius in solution, the recorded differences in the intensity of the scattered light reflect differences in the concentration of the aggregates in solution Assuming as 100% reference value the concentration of the aggregates of b-CN(1–25)4P in the presence of FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS 5003 CPP activity and supramolecular structure C Gravaghi et al Hydrodynamic radius of aggregates (nm) [Ca2+]o mmolỈL)1 b-CN(1–25)4P CPP DMV as1-CN(59–79)5P b-CN(1–25)0P Cluster sequence [Ca2+]o mmolỈL)1 b-CN(1–25)4P b-CN(1–25)0P Relative concentration of aggregates (%) 60 60 60 150 100 35 4.5 2.5 60 150 25 2.4 150 [Ca2+] mmol/L mmolỈL)1 Ca2+, which provides the highest scattered intensity (Table 1), the relative concentration of CPP DMV aggregates in the same solvent was 35%, although with a solute concentration six times higher than that of b-CN(1–25)4P, and that of as1-CN(59– 79)5P was only 4.5%, with the same total solute concentration The absence of Ca2+ caused a reduction in aggregation of b-CN(1–25)4P to only 25%, whereas no significant change in the relative percentage of aggregation was induced in b-CN(1–25)0P by the presence of Ca2+ (2.5% versus 2.4%) Of course, in each sample, aggregates are expected to coexist with disaggregated molecules, in a mole fraction depending on the physicochemical characteristics of the peptide However, the disaggregated fraction was shown to make a negligible contribution to the scattered intensity, less than 0.1% Laser light scattering measurements were also performed on CPP DMV (1280 lmolỈL)1), as1-CN(59– 79)5P (200 lmolỈL)1) and b-CN(1–25)4P (200 lmolỈL)1) as a function of Ca2+ concentration, in the same range of the Ca2+ uptake experiments reported in Fig 3, and the results are shown in Fig As the three CPPs form aggregated particles with the same hydrodynamic radius, as already reported, the differences in excess scattered intensity relative to the solvent, Ir ) 1, reflect the differences in the number of aggregates in solution The scattering intensity curves of b-CN(1–25)4P (Fig 5A) and CPP DMV (Fig 5B) present the same convex behavior, with a maximum at mmolỈL)1 Ca2+ It is surprising that the shapes closely correspond to those of the dose–biological response (Fig 3), showing that at mmolỈL)1 Ca2+, where the maximal biological activity is reached, there is the highest concentration of CPP aggregates In the 5004 β-CN(1-25)4P CPP DMV αS1-CN(59-79)5P 300 Scattered Intensity (relative units) Table Aggregative properties of CPPs.The data reported refer to experiments where the concentration of CPP was 1280 lmolỈL)1 for CPP DMV and 200 lmolỈL)1 for each other peptide in and mmolỈL)1 Ca2+ in phosphate-free KRH All data are referred to those for b-CN(1–25)4P, which provided the highest intensity of light scattering, assumed as 100% Fig Scattered intensity of CPPs as a function of Ca2+ concentration Scattered intensity curve for b-CN(1–25)4P (200 lmolỈL)1), for CPP DMV (1280 lmolỈL)1) and for as1-CN(59–79)5P (200 lmolỈL)1) Each value of scattered intensity is calculated in relative units, i.e with respect to the intensity scattered by the same amount of peptide as a full monomer solution case of as1-CN(59–79)5P (Fig 5C), the scattered intensity is always very low (as low as the biological effect) and shows a smooth increase of the number of aggregates with increasing Ca2+ content, again paralleling the similar small increase of the biological effect Discussion This work provides novel information regarding the ability of CPPs to enhance Ca2+ uptake by HT-29 cells, which have been shown to undergo differentiation in culture, and demonstrates that this biological effect depends on a particular type of CPP aggregation and the concentration of aggregates in solution For the first time, the supramolecular structural architecture of CPPs has been studied under experimental conditions that allow the viability in culture of cells such as differentiated HT-29 cells, and permit the expression by these cells of an enhanced uptake of extracellular Ca2+ Remarkably, the absence of phosphate ions (as KH2PO4) in the cell culture medium did not affect this biological effect, or cell viability, enabling us to explore the process of CPP aggregation (in the absence of any possible precipitation of calcium phosphate salts) by a laser light scattering technique Regarding the CPP-mediated enhancement of Ca2+ uptake, a relevant observation is the existence of an optimal CPP ⁄ Ca2+ ratio for the effect [4 mmolỈL)1 Ca2+ ⁄ 200 lmolỈL)1 b-CN(1–25)4P] This result, obtained in an experimental model consisting of in vitro cells, is in agreement with results obtained using animals or everted intestinal tissue [21–24] It is FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gravaghi et al noteworthy that the conditions we used, with Ca2+ concentrations up to mmolỈL)1, are close to those occurring in the intestinal lumen after a proper meal, where Ca2+ may reach a concentration of 3–4 mmolỈL)1 in rats and 7–8 mmolỈL)1 in humans [25] The modest Ca2+ uptake effect exerted on HT-29 cells by aS1-CN(59–79)5P as compared to the much more pronounced effect exerted by b-CN(1–25)4P is in line with the differences in the Fe2+ ⁄ 3+ absorption mediated by the two CPPs [26,27], possibly associated with different structural changes induced in the two CPPs by Fe2+ ⁄ 3+ (as well as Ca2+) binding [16,28,29] The set of laser light scattering experiments clearly demonstrated the occurrence of CPP self-aggregation in solution, with precise features (very rapid occurrence; 60 nm hydrodynamic radius; absolute need for concomitant presence of Ca2+ and CPP for optimal aggregation) At the same time, they also demonstrated that the ability to aggregate, in terms of dimension and concentration of aggregates, relied on the chemical structure of CPP, as the ‘cluster sequence’ pentapeptides not aggregate at all An explanation of these features can be given following a model of self-aggregation similar to that proposed by Horne for b-casein micelles [30,31], where the single monomers possess hydrophilic and hydrophobic regions, and hydrophobic interactions between monomers are important for the aggregation As CPPs are negatively charged, due to the presence of phosphorylated serine and glutamic acid residues, the repulsive interactions between monomers prevent their aggregation when they are dissolved in pure water, as we observed At higher ionic strengths, as in phosphate-free KRH, the effect of electrostatic repulsion is screened, and aggregation can take place, as we also observed In addition, calcium divalent counterions may facilitate the organization of peptides in the aggregates, as they can coordinate two charges belonging to different molecules, explaining the marked increase that we observed in the relative number of aggregates of b-CN(1–25)4P due just to the presence of Ca2+ The scarce propensity of as1-CN(59–79)5P to aggregate, in term of aggregate concentration, is most probably due to the additional phosphorylated serines present, providing more negative charges, and fewer hydrophobic residues [9] (Table 2) A strong contribution of repulsive interactions among monomers results in a higher proportion of monomeric forms, as compared to b-CN(1–25)4P The differences in the aggregation features and ability to elicit the [Ca2+]i rise effect of b-CN(1–25)4P and as1-CN(59–79)5P probably reflect the different and already described conformations of these CPPs [32,33] With regard to the different aggregation properties of CPP activity and supramolecular structure Table Synthetic CPP primary structures The ‘cluster sequence’ characteristic of all CPPs is underlined and indicated in bold characters S corresponds to phosphorylated serine (For additional details, see Ferraretto et al [18].) CPP Primary structure as1-CN(59–79)5P b-CN(1–25)4P b-CN(1–25)0P ‘Cluster sequence’ pentapeptide QMEAESISSSEEIVPNSVEQK(59–79) RELEELNVPGEIVESLSSSEESITR(1–25) RELEELNVPGEIVESLSSSEESITR(1–25) SSSEE b-CN(1–25)4P and b-CN(1–25)0P, b-CN(1–25)0P has a much lower number of negative charges than b-CN(1–25)4P, and an almost null coordination role due to Ca2+ Furthermore, b-CN(1–25)0P is known to assume a much more flexible and dynamic conformation in solution than b-CN(1–25)4P [32], which probably facilitates aggregation into bigger complexes In fact, the hydrodynamic radius of b-CN(1–25)0P is 150 nm versus the 60 nm of b-CN(1–25)4P However, the relative concentration of aggregates is about 2.5% that of b-CN(1–25)4P, regardless of the presence or absence of Ca2+ The two molecules aggregate but in a completely different manner, in terms of both size and concentration of aggregates, b-CN(1–25)4P aggregates exhibiting the Ca2+ uptake effect and b-CN(1– 25)0P not at all The absence of aggregation by the ‘cluster sequence’ is not surprising, as the presence of three phosphorylated serines and two glutamic acids accounts for such a strong negative charge that repulsive interactions prevail and prevent aggregation The most intriguing evidence provided by this investigation is the relationship between CPP aggregation and the biological effect on differentiated HT-29 cells As shown in Fig 5, the scattered intensity curves of b-CN(1–25)4P and CPP DMV at different Ca2+ concentrations exhibit the same convex behavior, with a maximum at mmolỈL)1 Ca2+, mimicking the profiles of the Ca2+ uptake effect (Fig 3) As the Ca2+ concentration increases from to mmolỈL)1, the concentration of aggregates increases, owing to the complexing power of Ca2+, but at higher contents, mmolỈL)1, the abundance of counterions leads to a higher number of phosphopeptide monomers undergoing direct interactions, preventing them from being involved in extensive aggregation In parallel, the biological effect rises from to mmolỈL)1 Ca2+, but decreases from to mmolỈL)1 Ca2+, indicating that it follows the concentration of aggregates This evidence suggests the notion that the aggregated forms are the active forms of a bioactive CPP such as b-CN(1–25)4P Further support for this notion comes from the finding that for formation FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS 5005 CPP activity and supramolecular structure C Gravaghi et al of the biologically active aggregates, the simultaneous presence of CPP and Ca2+ is needed while complexes are forming Presumably, the CPP aggregates formed in the absence of Ca2+, although exhibiting a hydrodynamic radius equal or similar to that of the Ca2+-containing aggregates (60 nm), are different from those formed in the presence of Ca2+ An additional relevant point concerns the role of phosphate in the CPP-mediated [Ca2+]i rise effect The removal of phosphate (as KH2PO4) from the buffer does not affect the biological effect, whereas the removal of phosphate from the serines totally abrogates it, emphasizing the fact that the role of serine-linked phosphate is essential to: (a) bind Ca2+; (b) induce correct aggregation of CPP; and (c) elicit the biological effect A final matter of discussion regards the possible relevance of our findings to the controversial issue [34,35] of whether CPPs enhance Ca2+ absorption at the intestinal level, thus improving Ca2+ bioavailability Investigations of this, performed on animals (rats, chicks, chickens) and humans, were based on the evidence that, in models of absorption such as everted sacs [21,36] and ligated segments of rat ileum [24,37,38], CPPs favor Ca2+ absorption, particularly in the presence of substances such as phosphate [36] that are capable of forming insoluble calcium salts This effect was attributed to the ability of CPPs to form complexes carrying ‘soluble’ calcium Our studies refer to a cell model, HT-29 cells differentiated in vitro Therefore, any extension to physiological situations in animals has to be done with extremely caution If we take this model as valid, the flux of Ca2+ from the extracellular milieu into the cytosol of HT-29 cells may mimic the Ca2+ flux from the intestinal lumen to the interior of enterocytes, particularly at the ileum level (passive absorption) The overall Ca2+ flux during intestinal absorption is in the mmolỈL)1 order of magnitude, whereas the observed increment of [Ca2+]i in HT-29 cells due to the CPP effect is in the range of about 50 nmolỈL)1 Whether this relatively small, although rapid, increase of [Ca2+]i is responsible for and sufficient to enable the passage of Ca2+ along the intestinal absorption route under physiological conditions is a difficult question that, at present, cannot be answered What can be said is that the [Ca2+]i rise effect does match the CPP-mediated enhanced Ca2+ absorption observed in the rat ileum sacs or ligated segments [21,24,36–38], substantiates the reports showing a positive role of CPP treatment on Ca2+ bioavailability in animals [22,24,39–44], and suggests the notion that CPPs not only maintain Ca2+ in an absorbable form but also interact with the plasma membranes of certain cells, facilitating Ca2+ uptake 5006 Concerning the conflicting results of human studies, some in favor of the efficacy of CPP treatment [45–48] and some not [49–51], it should be remembered that, according to our findings, the ability of CPPs to elicit the optimal biological effect relies on two critical conditions, the presence of Ca2–CPP aggregates in the correct conformation and concentration, and a suitable ratio between Ca2+ and CPP, this latter condition being in agreement with data determined in intestinal model studies [21,51] Examining the experimental protocols of the above-cited papers [45–51] it is hard to evaluate when (or whether) these critical conditions were fulfilled It is worth mentioning that we had evidence (unpublished results) that the ability of CPPs to elicit a transient rise in [Ca2+]i is acquired by HT-29 cells, as well as Caco-2 cells, upon differentiation (in other words, it is peculiar to the differentiated state of these intestinal-related cells), and is also exhibited by human osteoblasts in culture, suggesting that the CPP effect may be of more general significance in the modulation of Ca2+ uptake by cells It is the purpose of our current and future research to explore the molecular mechanism by which CPPs elicit a transient rise in [Ca2+]i in sensitive cells, as well as to set up and apply proper conditions to evaluate the use of CPPs as possible functional foods enhancing Ca2+ bioavailability Experimental procedures Cell culture media and all other reagents were purchased from Sigma (St Louis, MO, USA) Fetal bovine serum was from EuroClone Ltd (Wetherby, UK) Fura-2 acetoxymethyl ester, fura-2 pentasodium salt, and ionomycin (the last two compounds used only for calibration purposes) were obtained from Calbiochem (La Jolla, CA, USA) Casein phosphopeptides The CPP DMV preparation employed is a casein-derived hydrolysate (CE 90 CPP III; DMV International, Veghel, the Netherlands), comprising several components, each containing the characteristic CPP ‘cluster sequence’, with the following composition: 93.8% as dry matter; 96% purity; 10.8% total nitrogen content; 3.7% phosphorus content; nitrogen ⁄ phosphorus ratio 3.1; P ⁄ Ser ratio 0.85 mol ⁄ mol; average relative molecular mass 2500 This CPP mixture was determined to be Ca2+-free as already reported [17] The individual CPPs as1-CN(59–79)5P and b-CN(1–25)4P, the dephosphorylated form of b-CN(1–25)4P [b-CN(1–25)0P] and the ‘cluster sequence’ were synthetically produced by Primm (Milan, Italy), and characterized for purity as already reported [18] The primary structure of all the used synthetic FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gravaghi et al CPP activity and supramolecular structure peptides is shown in Table All CPPs and CPP derivatives were stored at ) 20 °C until use, when they were dissolved in double-distilled water in stock solutions (1000· concentrated, with respect to the final concentration) and eventually brought to neutrality with mmolỈL)1 NaOH Frickenhausen, Germany), were submitted to a h pulse with bromodeoxyuridine (BrdU), and BrdU incorporation into DNA was quantified by the chemiluminescent immunoassay (Roche Applied Science, Milan, Italy), following the manufacturer’s instructions Cell culture Isolation of brush border fraction and enzyme assays The colon carcinoma cell line HT-29 was obtained from the Istituto Zooprofilattico Sperimentale di Brescia (Brescia, Italy) In order to differentiate HT-29 cells, we used two different approaches: (a) to change the medium from highd-glucose DMEM to low-d-glucose RPMI supplemented with mmolỈL)1 l-glutamine, 0.1 mgỈL)1 streptomycin, · 105 L)1 penicillin and 0.25 mgỈL)1 amphotericin B, cells were cultured in RPMI medium until confluence, when they were subcultured for at least 10 passages; (b) substitution of glucose with galactose in DMEM medium ) these culture conditions guarantee a high degree of cell differentiation [52,53], as assessed by (i) measurement of the activity of ALP and sucrase-isomaltase, two well-known biochemical markers of intestinal cell differentiation, present on the brush border cell fraction (P2) isolated from the cell homogenates, (ii) measurement of their proliferation rate, and (iii) their fine morphology as analyzed by transmission electron microscopy Cell cultures were periodically checked for the presence of mycoplasma and were found to be free of contamination Cell viability, assessed by the Trypan blue exclusion test, and cell morphology, examined by optical microscopy, remained unaffected by treatment with each one of the used CPPs or CPP derivatives up to 40 mmolỈL)1 Electron microscopy Cells grown in DMEM, RPMI and in galactose-adapted DMEM were plated in 35 mm Petri dishes and allowed to grow until about 80% confluence, when they were fixed for 60 at room temperature with 2% glutaraldehyde in 0.1 m Sorensen phosphate buffer (pH 7.4), thoroughly rinsed with the same buffer, postfixed in 1% osmium tetraoxide (OsO4) in 0.1 m Sorensen phosphate buffer, dehydrated through an ascending series of ethanol, and embedded in araldite (Durcupan; Fluka, Milan, Italy) Ultrathin sections were obtained with an Ultracut ultramicrotome (Reichert Ultracut R-Ultramicrotome; Leika, Wien, Austria), and stained with uranyl acetate and lead citrate before examination using a Jeol CX100 electron microscope (Jeol, Tokyo, Japan) Cell proliferation assay Cells (1 · 104 cells per well), cultured in their medium in a Microtiter plate (96-well, Greiner bio-one; Cellstar, For the determination of ALP and sucrase-isomaltase activities, cells were seeded in 75 cm2 flasks and, after reaching 80–90% confluence, were harvested in ice-cold physiological saline, washed three times, pelleted by centrifugation at 105 000 g using a Beckman TL-100 (Beckman Coulter, Fullerton, CA, USA) rotor type TLA-100.3, and stored at ) 80 °C Cell subfractions, particularly the P2 subfraction enriched in brush borders, were prepared as described previously [54,55] The ALP assay was performed as previously described [56] on samples of 20–50 lg of P2 subfractions resuspended to a final volume of 50 lL The sucrase-isomaltase assay was performed following the one-step ultramicromethod [57] on P2 subfractions (about 20 lg of protein) resuspended to a final volume of 20 lL Results are expressed as m(mg protein))1, unit being defined as the enzyme activity that hydrolyzes lmole of substrate per minute The protein content was measured following the method of Lowry et al [58] [Ca2+]i measurement in cell populations The procedure described in our previous work [17] was employed Briefly, cells grown as a monolayer in a 25 cm2 flask in RPMI culture medium were detached with trypsin ⁄ EDTA, washed several times with KRH [containing (mmolỈL)1): NaCl 125.0, KCl 5.0, KH2PO4 1.2, CaCl2 2.0, MgSO4 1.2, glucose 6.0, and Hepes 25.0, pH 7.4), and loaded for 30 at 37 °C with lmolỈL)1 fura-2 acetoxymethyl ester and 2.5 lmolỈL)1 Pluronic F-127 in KRH The loaded cell suspension was rinsed extensively with KRH, and divided into aliquots comprising 0.5 · 106 cells Each aliquot was gently pelleted and resuspended in mL of KRH, and then transferred to a 37 °C thermostated cuvette in a Perkin-Elmer LS-50B spectrofluorimeter (Perkin-Elmer, Beaconsfield, UK) This fura-2-loaded cell suspension was continuously stirred, and concomitantly submitted to excitation at 343 nm, the fluorescence intensity being recorded at 485 nm As fura-2 fluorescence increases with increasing [Ca2+]i at these wavelength settings, the changes in fluorescence intensity reflected the changes in [Ca2+]i concentration [59] CPP was administered to cell suspension at the final chosen concentration, and at the end of each experiment a calibration was performed [17] The peak of [Ca2+]i increase was calculated as the difference between the [Ca2+]i values recorded after and before (basal value) FEBS Journal 274 (2007) 4999–5011 ª 2007 The Authors Journal compilation ª 2007 FEBS 5007 CPP activity and supramolecular structure C Gravaghi et al CPP administration, and was expressed as percentage of the basal value or in absolute terms (nmolỈL)1) Under these conditions, the duration of the experiments was less than 10 min, including a 1–2 interval between the addition of Ca2+ and that of CPP In this period of time, cells maintained full viability As a control, [Ca2+]i concentrations after ionomycin treatment [59] were also measured Experiments with increasing extracellular Ca2+ concentrations In the experiments performed in the presence of [Ca2+]o higher than mmolỈL)1, fura-2-loaded cell pellets were suspended, immediately before starting the experiment, in phosphate-free KRH containing (mmolỈL)1) 140.0 NaCl, 5.0 KCl, 0.55 MgCl2, 6.0 glucose and 10.0 Hepes, adjusted to pH 7.4, to prevent any possible precipitation of calcium phosphate Then, CaCl2 was added in order to obtain the desired final Ca2+ concentrations Characterization of CPP aggregation by laser light scattering The aggregative properties of CPPs were studied by laser light scattering Aliquots of different CPPs, dissolved in pure water as concentrated stocks, were diluted to the final concentrations in KRH or in phosphate-free KRH, containing the appropriate Ca2+ concentration, matching the experimental conditions used to follow the CPP biological effect The absence of any calcium phosphate precipitation was a prerequisite for light scattering measurement The samples were transferred in an appropriate measuring cell, and quasielastic laser light scattering measurements were carried out on a standard apparatus equipped with a BI9K Digital correlator (Brookhaven Instruments Co., Holtsville, NY, USA) [60] The light source was an argon ion laser operating on the 514 nm green line (Lexel, Fremont, CA, USA) Both independent static and dynamic laser light scattering measurements were performed on the same samples at room temperature If molecules undergo aggregation in solution, laser light scattering immediately reveals the presence of aggregates, recognizing both the dimension (hydrodynamic radius) and the concentration of the aggregated particles Static measurements provide combined information about the average molecular mass and the concentration of macromolecules in solution The measured quantity is the average light intensity scattered by the solution relative to that scattered by the solvent All of the solvents used in our experiments (water, phosphate-free KRH and KRH) showed the same extremely low scattered intensity within experimental errors The excess of scattered light due to the presence of CPP, Ir ) ¼ (ICPPsolution ) Isolvent) ⁄ Isolvent is proportional to both the average molecular mass and the concentration of CPP particles in solution according to the equation: 5008  2 X  2 dn cn dn Ir À ¼ A Mn ¼ A c c dc dc c ð1Þ where A is a calibration constant, dn ⁄ dc is the refractive index increment of the solution, c is the CPP concentration (gỈmL)1), cn is the concentration of CPP forming particles of molecular mass Mn, and is the average molecular mass of the CPP particles in solution Independently, dynamic measurements yield information about the diffusion coefficient D of particles in solution, and hence their hydrodynamic radius, RH, via the Stokes–Einstein relation: Dẳ kB T 6pgRH 2ị where kB is Boltzmanns constant, T is the absolute temperature, and g is the viscosity of the solvent [60,61] If particles of different dimensions are present in solution, they can be resolved, as their contribution to the measured correlation function has a characteristic decay time proportional to their dimension Therefore, the availability of both static and dynamic laser light scattering measurements enables us to decouple information about the average mass and relative concentration of CPP aggregates in solution Statistical analysis The data reported in Figs and are expressed as mean values ± SD Statistically significant differences between two mean values were established by Student’s t-test, and two independent population t-tests, performed with origin 6.0 (Origin Lab Corporation, Northampton, MA, USA) (a P-value < 0.05 was considered significant) Acknowledgements This work was supported in part by the EU FAIR Programme Project CT98-3077 [Casein phosphopeptide (CPP): Nutraceutical ⁄ functional food ingredients for food and pharmaceutical applications] and by Fondazione Romeo ad Enrica Invernizzi (CPP: role in the calcium intestinal absorption and its utilization A perspective study on their possible usage as nutraceuticals or functional food to favour calcium bioavailability) We thank Professor Mario Corti for helpful reading and discussing the 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(200 1) Effect of caseinphosphopeptides on inhibition of calcium intestinal absorption due to phosphate Nutr Res 21, 649–656 37 Lee YS, Noguchi T & Naito H (198 3) Intestinal absorption of calcium in. .. (199 1) In? ??uence of casein phosphopeptides and lactulose on intestinal calcium absorption in adult female rats Lait 71, 173– 180 42 Kopra N, Scholz-Ahrens KE & Barth CA (199 2) Effect of casein phosphopeptides