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

Calcium phosphate nanoclusters or complexes were also prepared and physicochemically character-ized using CPPs, namely b-CN1–254P and b-CN1– 425P, corresponding to the first 25 or 42 amin

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 Tettamanti1and Anita Ferraretto1

1 Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy

2 DMU, Department of Human Morphology, University of Milan, Italy

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,

Keywords

Ca 2+ 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)1Ca2+

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.

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

correspond-ing 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

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 supramolecular structure of

CPP–Ca2+complexes is required for the observed

bio-logical 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

occur-rence 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

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 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 sucrase-isomaltase, respectively), whereas galactose-adapted cells showed a marked increase of both ALP (830 ± 12 mUÆmg)1 protein) and sucrase-isomaltase (270 ± 20 mUÆmg)1protein) 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, 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

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

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

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)1in the presence of phosphate (trace a) and 70 nmolÆL)1in the absence of phosphate (trace b), whereas the increments due to CPP were 25 nmolÆL)1and 22 nmolÆL)1, respec-tively; for 2 mmolÆL)1 [Ca2+]o and 100 lmolÆL)1 b-CN(1–25)4P, the basal Ca2+ concentration was

72 nmolÆL)1 (trace c) and 84 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 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+]owas raised to 6 mmolÆL)1and the three different preparations of CPP, each at different con-centrations, were employed With b-CN(1–25)4P and

0 250 500 750

1000

A

B

GALACTOSE RPMI

DMEM

GALACTOSE RPMI

DMEM

*

*

ALP sucrase

0 50

100

*

*

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.

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)1CPP 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-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-containfind-ing 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

0 50

100

[β-CN(1-25)4P]

50μmol/L

0

100 50

*

*

[Ca2+]o 2mmol/L

0 50 100

Ionomycin Ionomycin

a

[β-CN(1-25)4P]

100μmol/L

Time (s)

200

*

*

[Ca2+]o 4mmol/L

KRH (containing phosphate) phosphate-free KRH

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 Ca 2+ 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 [Ca 2+ ] 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)1b-CN(1–25)4P (arrow) in KRH (trace a), and in phosphate-free KRH (trace b), and the representative traces relative to 100 lmolÆL)1b-CN(1–25)4P (arrow) in KRH (trace c), in phosphate-free KRH (trace d) at 2 mmolÆL)1extracellular Ca2+ concentration, are shown The vertical scale indicates fluorescent intensity at 485 nm emission wavelength after excitation at 343 nm.

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

of 10 times that of the pure solvent, indicating the

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

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 dis-similar to that of pure solvent

Dynamic light scattering experiments showed (Table 1) that the three CPPs, b-CN(1–25)4P, as1 -CN(59–79)5P and CPP DMV, dissolved in 4 mmolÆL)1

Ca2+ phosphate-free KRH, formed aggregated struc-tures with the same hydrodynamic radius (RH¼

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 (RH¼ 150 ± 4 nm), regardless of the presence or absence of Ca2+

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

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

0

150

300

A

B

C

0

150

300

0

150

300

[ β-CN(1-25)4P]

200 μmol/L

1280 μmol/L

960 μmol/L

640 μmol/L

320 μmol/L

200 μmol/L

150 μmol/L

100 μmol/L

50 μmol/L

150 μmol/L

100 μmol/L

50 μmol/L

] i

[CPP DMV]

[Ca 2+ ] o mmol/L

[αs1 -CN(59-79)5P]

4

Fig 3 CPP bioactivity is related to extracellular Ca 2+ 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 a s1 -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 [Ca 2+ ]ipeak 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 Ca 2+ concentration All values are

signi-ficantly different from each other (P < 0.05).

0 25 50

β-CN(1-25)0P β-CN(1-25)4P

β I-1r

1 2 3 4 5

Fig 4 Excess scattered intensity relative to the solvent, I r ) 1, for:

1, b-CN(1–25)4P in phosphate-free KRH containing 4 mmolÆL)1

Ca 2+ 2, b-CN(1–25)4P in phosphate-free KRH without Ca 2+ ; 3, b-CN(1–25)4P prepared in phosphate-free KRH without Ca 2+ fol-lowed by addition of 4 mmolÆL)1 Ca2+; 4, b-CN(1–25)0P in phos-phate-free KRH containing 4 mmolÆL)1 Ca 2+ ; 5, b-CN(1–25)0P in phosphate-free KRH without Ca 2+ (for all solvents, phosphate-free KRH with or without 4 mmolÆL)1Ca2+, the same very small scat-tered intensity was measured).

4 mmolÆL)1Ca2+, 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

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%

Laser light scattering measurements were also

performed on CPP DMV (1280 lmolÆL)1), as1-CN(59–

79)5P (200 lmolÆL)1) and b-CN(1–25)4P (200

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

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 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 concentraaggrega-tion of aggregates in soluaggrega-tion 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 such as differentiated HT-29 cells, and permit the 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 precipitaaggrega-tion 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 mmolÆL)1

Ca2+⁄ 200 lmolÆL)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

0

150 300

Scattered Intensity (relative units)

[Ca 2+ ] mmol/L

β-CN(1-25)4P CPP DMV

αS1 -CN(59-79)5P

Fig 5 Scattered intensity of CPPs as a function of Ca 2+ 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.

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 Ca 2+ 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%.

Hydrodynamic radius

of aggregates (nm)

Relative concentration

of aggregates (%) [Ca2+] o 4 mmolÆL)1

[Ca2+] o 0 mmolÆL)1

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

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

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

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

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 soluconforma-tion 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)1Ca2+, 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

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].)

a s1 -CN(59–79)5P QMEAESISSSEEIVPNSVEQK(59–79) b-CN(1–25)4P RELEELNVPGEIVESLSSSEESITR(1–25) b-CN(1–25)0P RELEELNVPGEIVESLSSSEESITR(1–25)

‘Cluster sequence’

pentapeptide

SSSEE

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

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

rel-evance 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

evi-dence 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 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 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

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 [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

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 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 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

It is the purpose of our current and future research

to explore the molecular mechanism by which CPPs elicit a transient rise in [Ca2+]iin 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 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)

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 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;

average relative molecular mass 2500 This CPP mixture was

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

peptides is shown in Table 2 All CPPs and CPP derivatives

double-distilled water in stock solutions (1000·

concen-trated, with respect to the final concentration) and

Cell culture

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

cells were cultured in RPMI medium until confluence, when

they were subcultured for at least 10 passages; (b)

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

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

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 min 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

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-Ultraultra-microtome; 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

a Microtiter plate (96-well, Greiner bio-one; Cellstar,

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

Isolation of brush border fraction and enzyme assays

For the determination of ALP and sucrase-isomaltase

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 pre-viously [54,55] The ALP assay was performed as prepre-viously 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

the enzyme activity that hydrolyzes 1 lmole of substrate per minute The protein content was measured following the method of Lowry et al [58]

[Ca2+]imeasurement in cell populations The procedure described in our previous work [17] was

flask in RPMI culture medium were detached with

The loaded cell suspension was rinsed extensively with

Each aliquot was gently pelleted and resuspended in 2 mL

(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

changes in fluorescence intensity reflected the changes in

sus-pension at the final chosen concentration, and at the end of each experiment a calibration was performed [17] The peak

CPP administration, and was expressed as percentage of the

conditions, the duration of the experiments was less than

10 min, including a 1–2 min interval between the addition of

after ionomycin treatment [59] were also measured

Experiments with increasing extracellular Ca2+

concentrations

suspended, immediately before starting the experiment, in

adjusted to pH 7.4, to prevent any possible precipitation

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,

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

sam-ples 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

aggre-gated particles Static measurements provide combined

information about the average molecular mass and the

con-centration of macromolecules in solution The measured

quantity is the average light intensity scattered by the

solu-tion relative to that scattered by the solvent All of the

sol-vents used in our experiments (water, phosphate-free KRH

and KRH) showed the same extremely low scattered

inten-sity within experimental errors The excess of scattered

molecu-lar mass and the concentration of CPP particles in solution

according to the equation:

dc

 2

dc

 2

index increment of the solution, c is the CPP

aver-age molecular mass of the CPP particles in solution Independently, dynamic measurements yield information about the diffusion coefficient D of particles in solution,

Stokes–Einstein relation:

D¼ kBT 6pgRH

ð2Þ

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 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 1 and 2 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

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 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

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