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
1 Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy 2 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 6 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 stud- ied 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 4 mmolÆL)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 concomi- tant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggrega- tion 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 4 mmolÆL)1 Ca2+.
It is known that milk is an excellent source of bioavail- able 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 : 1 [6]. The casein micelles, of about 100 nm radius, are stable structures composed of hundreds of smaller aggregates, named calcium phosphate nanocl- usters, 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 contain- ing 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 character- ized 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.
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corresponding to the sequence 59–79 of aS1-casein [8,9,14–16].
those providing the mentioned biological effect of CPP was of central importance.
Results
to precise
fragments of
supramolecular
calcium phosphate–CPP aggregation
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 correspond- ing the b-CN(1–25)4P sequence, clarified that a well-defined primary structure is required for the bioactive response [18]. This struc- ture 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, suggest- ing that a particular structure of CPP–Ca2+ complexes is required for the observed bio- logical effect in vitro, by analogy with the relationship between as nanoclusters and the capacity to bind and maintain calcium in a bioavailable form.
The present
sucrase-isomaltase,
(KRH)
laminae
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 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 occur- these conditions of a supramolecular rence under 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
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 struc- tural 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 mmolÆL)1) do not present signs of spontaneous dif- ferentiation towards intestinal-like cells [19]. Instead, when the culture medium is switched to RPMI, with low d-glucose concentration (13.9 mmolÆL)1), or to a DMEM medium with galactose gradually substituting for glucose, HT-29 cells undergo a process of intesti- nal-like differentiation [20]. On this basis, HT-29 cells were cultured in RPMI (low d-glucose) or galactose- containing 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-micro- scopic 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 mUÆmg)1 protein for ALP, and 80.7 ± 9.1 versus 77.8 ± 8.3 mUÆmg)1 pro- tein for respectively), whereas galactose-adapted cells showed a marked increase of both ALP (830 ± 12 mUÆmg)1 protein) and sucrase- isomaltase (270 ± 20 mUÆmg)1 protein). The prolifera- tion rate (Fig. 1B) of cells cultured in RPMI and galactose-adapted medium markedly decreased as com- pared 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 desmo- somes, and galactose-adapted cells display all the features observed in RPMI cells, with, in addition, surrounded by intracellular characteristic numerous and well-developed small microvilli. All of these findings indicate that HT-29 cells grown in RPMI or galactose-containing medium undergo a intestinal-like differentiation, remarkable process of confirming previous data [19,20]. From both the quantitative and qualitative points of view, both
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ALP sucrase
1000A
*
750
500
/
*
n i e t o r p g m U m
250
0
DMEM
RPMI
GALACTOSE
B
)
100
%
*
*
50
( n o i t a r o p r o c n
i
U d r B
0
DMEM
RPMI
GALACTOSE
C
D
E
Fig. 1. HT-29 cell differentiation. (A) ALP (white bars) and sucrase (black bars) enzyme activities of DMEM (undifferenti- ated), RPMI and galactose-adapted (differen- tiated) cells. (B) Proliferation rate as determined by BrdU incorporation in the three cell populations expressed as percent- age 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). Aster- isks indicate significantly different values (P < 0.05) from DMEM.
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 experi- ments were performed by culturing cells in RPMI medium.
for 2 mmolÆL)1
and 84 nmolÆL)1
(trace c)
values, were the same regardless of the presence or absence of phosphate. In more detail (Fig. 2C,D), for 2 mmolÆL)1 [Ca2+]o and 50 lmolÆL)1 b-CN(1–25)4P, the basal Ca2+ concentration was 100 nmolÆL)1 in the presence of phosphate (trace a) and 70 nmolÆL)1 in the absence of phosphate (trace b), whereas the increments due to CPP were 25 nmolÆL)1 and 22 nmolÆL)1, respec- [Ca2+]o and 100 lmolÆL)1 tively; the basal Ca2+ concentration was b-CN(1–25)4P, 72 nmolÆL)1 (trace d), whereas the increments due to CPP were 48 nmolÆL)1 and 47 nmolÆL)1, respectively, i.e. the same regardless of the presence or absence of phosphate in the buffer.
indicating that
Similar results were obtained with the CPP DMV free mixture and as1-CN(59–79)5P, 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 6 mmolÆL)1 and the three different preparations of CPP, each at different con- centrations, were employed. With b-CN(1–25)4P and
To investigate the effect of CPP in increasing the extracellular free Ca2+ concentration ([Ca2+]o) in the buffer solution, while avoiding the possible precipita- tion of insoluble calcium phosphate salts, which would affect biological and laser light scattering measure- ments, we first explored whether the presence of phos- phate was necessary for the biological effect of CPP. To this end, a first dose–response set of experiments at [Ca2+]o higher than 2 mmolÆL)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 lmolÆL)1) and in the presence of 2 or 4 mmolÆL)1 [Ca2+]o, expressed as percentage of the basal
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KRH (containing phosphate) phosphate-free KRH
A
100
B 100
[Ca2+]o 2mmol/L
[Ca2+]o 4mmol/L
*
*
*
*
e s a e r c n
i
50
50
) e u l a v l a s a b n o %
(
m u i c l a c k a e p
0
0
50
50
100 [β-CN(1-25)4P] μmol/L
100 [β-CN(1-25)4P] μmol/L
C
600
D 600
a b
c d
Ionomycin
[β-CN(1-25)4P] 100 μmol/L
[β-CN(1-25)4P] 50 μmol/L
Ionomycin
400
400
s t i n u y r a r t i b r a e c n e c s e r o u l F
200
200
0
0
200
100
100
200 Time (s)
Fig. 2. 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 lmolÆL)1) and at two different extracellular Ca2+ concentrations, 2 mmolÆL)1 (A) and 4 mmolÆL)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 lmolÆL)1 b-CN(1–25)4P (arrow) in KRH (trace a), and in phosphate- free KRH (trace b), and the representative traces relative to 100 lmolÆL)1 b-CN(1–25)4P (arrow) in KRH (trace c), in phosphate-free KRH (trace d) at 2 mmolÆL)1 extracellular Ca2+ concentration, are shown. The vertical scale indicates fluorescent intensity at 485 nm emission wavelength after excitation at 343 nm.
should be remembered that
It
tigated range of Ca2+ concentration (2–6 mmolÆL)1), was recorded at 6 mmolÆL)1 [Ca2+]o. Third, no signifi- cant 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 find- ing 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. the ‘cluster sequence’ is completely unable to elicit the increase in [Ca2+]i [18].
CPP DMV mixture, the highest biological effects were observed at 4 mmolÆL)1 [Ca2+]o (Fig. 3A,B), the opti- mal effect being obtained at 200 lmolÆL)1 b-CN(1– 25)4P and 1280 lmolÆL)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 inves-
Preliminary laser light scattering experiments showed that an aqueous solution of b-CN(1–25)4P, as well as
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300
[β-CN(1-25)4P]
A
50
1 2 3 4 5
150
200 μmol/L 150 μmol/L 100 μmol/L 50 μmol/L
r
25
1 - I
0
B
[CPP DMV]
300
0
β β-CN(1-25)4P
β-CN(1-25)0P
e s a e r c n i
i
150
1280 μmol/L 960 μmol/L 640 μmol/L 320 μmol/L
] + 2 a C
) e u l a v l a s a b n o
%
(
[ k a e p
0
[α
300
C
s1-CN(59-79)5P]
Fig. 4. Excess scattered intensity relative to the solvent, Ir ) 1, for: 1, b-CN(1–25)4P in phosphate-free KRH containing 4 mmolÆL)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+ fol- lowed by addition of 4 mmolÆL)1 Ca2+; 4, b-CN(1–25)0P in phos- phate-free KRH containing 4 mmolÆL)1 Ca2+; 5, b-CN(1–25)0P in phosphate-free KRH without Ca2+ (for all solvents, phosphate-free KRH with or without 4 mmolÆL)1 Ca2+, the same very small scat- tered intensity was measured).
150
200 μmol/L 150 μmol/L 100 μmol/L 50 μmol/L
0
2
6
4 [Ca2+]ommol/L
Fig. 3. CPP bioactivity is related to extracellular Ca2+ and peptide concentration. The data were collected after administering to fura- 2-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 cor- responds 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 signi- ficantly different from each other (P < 0.05).
of aggregation corresponded to the duration of the experimental manipulations (pipetting, mixing, etc.), i.e. a few seconds. This indicates that the process of is very rapid. b-CN(1– aggregation, when it occurs, 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 dis- similar to that of pure solvent. scattering light
experiments
Dynamic
that
regardless of
showed the three CPPs, b-CN(1–25)4P, as1- (Table 1) CN(59–79)5P and CPP DMV, dissolved in 4 mmolÆL)1 Ca2+ phosphate-free KRH, formed aggregated struc- (RH ¼ tures with the same hydrodynamic radius 60 ± 2 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 ± 4 nm), the presence or absence of Ca2+.
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 con- trast, 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 indicating the of 10 times that of the pure solvent, occurrence of some aggregation. An additional four- fold increase of the scattered light occurred when the solvent contained 4 mmolÆL)1 [Ca2+]o, whereas addi- tion 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
Concerning the three CPPs with the same hydro- dynamic radius in solution, the recorded differences in the intensity of the scattered light do reflect differences the aggregates in solution. in the concentration of Assuming as 100% reference value the concentration of the aggregates of b-CN(1–25)4P in the presence of
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β-CN(1-25)4P CPP DMV α S1-CN(59-79)5P
Table 1. Aggregative properties of CPPs.The data reported refer to experiments where the concentration of CPP was 1280 lmolÆL)1 for CPP DMV and 200 lmolÆL)1 for each other peptide in 4 and 0 mmolÆL)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%.
150
Hydrodynamic radius of aggregates (nm)
Relative concentration of aggregates (%)
) s t i n u e v i t a l e r (
y t i s n e t n I d e r e t t a c S
0
100 35
2
6
4 [Ca2+] mmol/L
60 60 60 150 0
4.5 2.5 0
25
60 150
2.4
[Ca2+]o 4 mmolÆL)1 b-CN(1–25)4P CPP DMV as1-CN(59–79)5P b-CN(1–25)0P Cluster sequence [Ca2+]o 0 mmolÆL)1 b-CN(1–25)4P b-CN(1–25)0P
Fig. 5. Scattered intensity of CPPs as a function of Ca2+ concentra- tion. Scattered intensity curve for b-CN(1–25)4P (200 lmolÆL)1), for CPP DMV (1280 lmolÆL)1) and for as1-CN(59–79)5P (200 lmolÆL)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.
the
relative
case of as1-CN(59–79)5P (Fig. 5C), the scattered inten- sity is always very low (as low as the biological effect) and shows a smooth increase of the number of aggre- gates with increasing Ca2+ content, again paralleling the similar small increase of the biological effect.
Discussion
4 mmolÆL)1 Ca2+, which provides the highest scattered intensity (Table 1), 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 con- centration. 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 pres- ence of Ca2+ (2.5% versus 2.4%). Of course, in each sample, aggregates are expected to coexist with dis- aggregated 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 inten- sity, less than 0.1%.
light
Laser
b-CN(1–25)4P
(200 lmolÆL)1)
This work provides novel information regarding the ability of CPPs to enhance Ca2+ uptake by HT-29 cells, which have been shown to undergo differentia- tion in culture, and demonstrates that this biological effect depends on a particular type of CPP aggrega- tion and the concentration of aggregates in solution. For the first time, the supramolecular structural archi- tecture of CPPs has been studied under experimental conditions that allow the viability in culture of cells the such as differentiated HT-29 cells, and permit expression by these cells of an enhanced uptake of extracellular Ca2+. Remarkably, the absence of phos- phate 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 aggrega- tion (in the absence of any possible precipitation of calcium phosphate salts) by a laser light scattering technique.
b-CN(1–25)4P].
This
scattering measurements were also performed on CPP DMV (1280 lmolÆL)1), as1-CN(59– (200 and 79)5P lmolÆL)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. 5. As the three CPPs form aggregated particles with the same hydrodynamic radius, as already reported, the differ- ences in excess scattered intensity relative to the sol- vent, 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 4 mmolÆL)1 Ca2+. It is surprising that the shapes closely correspond to those of the dose–biological response (Fig. 3), showing that at 4 mmolÆL)1 Ca2+, where the maximal biological activity is reached, there is the highest concentration of CPP aggregates. In the
Regarding the CPP-mediated enhancement of Ca2+ uptake, a relevant observation is the existence of an optimal CPP ⁄ Ca2+ ratio for the effect [4 mmolÆL)1 Ca2+ ⁄ 200 lmolÆL)1 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
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Table 2. Synthetic CPP primary structures. The ‘cluster sequence’ characteristic of all CPPs is underlined and indicated in bold charac- ters. 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’
QMEAESISSSEEIVPNSVEQK(59–79) RELEELNVPGEIVESLSSSEESITR(1–25) RELEELNVPGEIVESLSSSEESITR(1–25) SSSEE
pentapeptide
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 conforma- tion in solution than b-CN(1–25)4P [32], which proba- bly 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 do aggregate but in a completely different manner, in terms of both size and concentration of aggregates, b-CN(1–25)4P aggre- gates 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 repul- sive interactions prevail and prevent aggregation.
The most intriguing evidence provided by this inves- tigation 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+ concen- trations exhibit the same convex behavior, with a maxi- mum at 4 mmolÆL)1 Ca2+, mimicking the profiles of the Ca2+ uptake effect (Fig. 3). As the Ca2+ concen- tration increases from 2 to 4 mmolÆL)1, the concentra- tion of aggregates increases, owing to the complexing power of Ca2+, but at higher contents, 6 mmolÆL)1, the abundance of counterions leads to a higher number of phosphopeptide monomers undergoing direct inter- actions, preventing them from being involved in exten- sive aggregation. In parallel, the biological effect rises from 2 to 4 mmolÆL)1 Ca2+, but decreases from 4 to 6 mmolÆL)1 Ca2+, indicating that it follows the concen- tration of aggregates. This evidence suggests the notion that the aggregated forms are the active forms of a bio- active CPP such as b-CN(1–25)4P. Further support for this notion comes from the finding that for formation
noteworthy that the conditions we used, with Ca2+ concentrations up to 6 mmolÆL)1, are close to those occurring in the intestinal lumen after a proper meal, where Ca2+ may reach a concentration of 3–4 mmolÆL)1 in rats and 7–8 mmolÆL)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 in the Fe2+ ⁄ 3+ absorption with the differences 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 occur- rence; 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’ pentapep- tides do not aggregate at all. An explanation of these features can be given following a model of self-aggre- gation 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 mono- mers 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 elec- trostatic 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 nega- tive charges, and fewer hydrophobic residues [9] (Table 2). A strong contribution of repulsive interac- tions 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 the different and as1-CN(59–79)5P probably reflect already described conformations of these CPPs [32,33]. With regard to the different aggregation properties of
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CPP activity and supramolecular structure
studies
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 hydrody- namic radius equal or similar to that of the Ca2+-con- taining 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-medi- ated [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 to: the role of serine-linked phosphate is essential (a) bind Ca2+; (b) induce correct aggregation of CPP; and (c) elicit the biological effect.
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 con- ditions, the presence of Ca2–CPP aggregates in the cor- rect conformation and concentration, and a suitable ratio between Ca2+ and CPP, this latter condition being in agreement with data determined in intestinal model [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 evi- dence (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 mod- ulation of Ca2+ uptake by cells.
rat
[21,36] and ligated segments of
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
Casein phosphopeptides
this
[22,24,39–44], and suggests
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 acetoxy- methyl ester, fura-2 pentasodium salt, and ionomycin (the last two compounds used only for calibration purposes) were obtained from Calbiochem (La Jolla, CA, USA).
A final matter of discussion regards the possible rel- evance of our findings to the controversial issue [34,35] of whether CPPs enhance Ca2+ absorption at the level, thus improving Ca2+ bioavailability. intestinal Investigations of this, performed on animals (rats, chicks, chickens) and humans, were based on the evi- in models of absorption such as everted dence that, sacs 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 mmolÆL)1 order of mag- nitude, whereas the observed increment of [Ca2+]i in HT-29 cells due to the CPP effect is in the range of about 50 nmolÆL)1. Whether 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 condi- tions 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 show- ing a positive role of CPP treatment on Ca2+ bioavail- ability in animals 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.
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The CPP DMV preparation employed is a casein-derived hydrolysate (CE 90 CPP III; DMV International, Veghel, the Netherlands), comprising several components, each con- taining 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
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CPP activity and supramolecular structure
Cell culture
Isolation of brush border fraction and enzyme assays
Frickenhausen, Germany), were submitted to a 2 h pulse with bromodeoxyuridine (BrdU), and BrdU incorporation into DNA was quantified by the chemiluminescent immu- noassay (Roche Applied Science, Milan, Italy), following the manufacturer’s instructions. peptides is shown in Table 2. All CPPs and CPP derivatives were stored at ) 20 (cid:2)C until use, when they were dissolved in double-distilled water in stock solutions (1000· concen- trated, with respect to the final concentration) and eventu- ally brought to neutrality with 1 mmolÆL)1 NaOH.
[Ca2+]i measurement in cell populations
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 high- d-glucose DMEM to low-d-glucose RPMI supplemented with 2 mmolÆL)1 l-glutamine, 0.1 mgÆL)1 streptomycin, 1 · 105 UÆL)1 penicillin and 0.25 mgÆL)1 amphotericin B, cells were cultured in RPMI medium until confluence, when they were subcultured for at least 10 passages; (b) substitu- tion of glucose with galactose in DMEM medium ) these culture conditions guarantee a high degree of cell differenti- ation [52,53], as assessed by (i) measurement of the activity of ALP and sucrase-isomaltase, two well-known biochemi- cal markers of intestinal cell differentiation, present on the brush border cell fraction (P2) isolated from the cell homo- genates, (ii) measurement of their proliferation rate, and (iii) their fine morphology as analyzed by transmission elec- tron microscopy. For the determination of ALP and sucrase-isomaltase activ- ities, 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 (cid:2)C. Cell subfractions, particularly the P2 subfraction enriched in brush borders, were prepared as described pre- viously [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-iso- maltase assay was performed following the one-step ultra- micromethod [57] on P2 subfractions (about 20 lg of protein) resuspended to a final volume of 20 lL. Results are expressed as mUÆ(mg protein))1, 1 unit being defined as the enzyme activity that hydrolyzes 1 lmole of substrate per minute. The protein content was measured following the method of Lowry et al. [58].
Electron microscopy
Cell cultures were periodically checked for the presence of mycoplasma and were found to be free of contamina- tion. 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 mmolÆL)1.
(pH 7.4),
Cell proliferation assay
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 min at room temperature with 2% glutaraldehyde in 0.1 m Sorensen phosphate buffer thoroughly rinsed with the same buffer, postfixed in 1% osmium tetra- oxide (OsO4) in 0.1 m Sorensen phosphate buffer, dehy- drated through an ascending series of ethanol, and embedded in araldite (Durcupan; Fluka, Milan, Italy). Ultrathin sections were obtained with an Ultracut ultra- microtome (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).
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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 tryp- sin ⁄ EDTA, washed several times with KRH [containing (mmolÆL)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 min at 37 (cid:2)C with 5 lmolÆL)1 fura-2 acet- oxymethyl ester and 2.5 lmolÆL)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 2 mL of KRH, and then transferred to a 37 (cid:2)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 sub- mitted 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 sus- pension 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) Cells (1 · 104 cells per well), cultured in their medium in a Microtiter plate (96-well, Greiner bio-one; Cellstar,
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CPP activity and supramolecular structure
c < M > c ð1Þ Ir (cid:2) 1 ¼ A Mn ¼ A (cid:2) (cid:3)2 dn dc (cid:2) (cid:3)2 dn dc X cn c
Experiments with increasing extracellular Ca2+ concentrations
CPP administration, and was expressed as percentage of the basal value or in absolute terms (nmolÆL)1). Under these conditions, the duration of the experiments was less than 10 min, including a 1–2 min interval between the addition of Ca2+ and that of CPP. In this period of time, cells main- [Ca2+]i concentrations tained full viability. As a control, after ionomycin treatment [59] were also measured.
D ¼
ð2Þ
kBT 6pgRH
their hydrodynamic where A is a calibration constant, dn ⁄ dc is the refractive
index increment of the solution, c is the CPP concentra-
tion (gÆmL)1), cn is the concentration of CPP forming
particles of molecular mass Mn, and
In the experiments performed in the presence of [Ca2+]o higher than 2 mmolÆL)1, fura-2-loaded cell pellets were suspended, immediately before starting the experiment, in phosphate-free KRH containing (mmolÆL)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
to their dimension. Therefore,
Statistical analysis
where kB is Boltzmann’s constant, T is the absolute temper- ature, and g is the viscosity of the solvent [60,61]. If parti- cles of different dimensions are present in solution, they can be resolved, as their contribution to the measured cor- relation function has a characteristic decay time propor- tional 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.
Acknowledgements
This work was supported in part by the EU FAIR Programme Project CT98-3077 [Casein phosphopep- tide (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 nutraceuti- cals or functional food to favour calcium bioavailabil- ity). We thank Professor Mario Corti for helpful reading and discussing the manuscript.
References
The data reported in Figs 1 and 2 are expressed as mean values ± SD. Statistically significant differences between t-test, two mean values were established by Student’s 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).
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CPP activity and supramolecular structure
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