Food Chemistry 166 (2015) 492–497

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Analytical Methods

Metal ions bound to the human milk immunoglobulin A:Metalloproteomic approach

http://dx.doi.org/10.1016/j.foodchem.2014.06.0400308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: Institute of Biosciences, Department of Chem-istry and Biochemistry, São Paulo State University-UNESP, Distrito de Rubião Júnior,18618-970 Botucatu, SP, Brazil. Tel.: +55 (14) 3880 0609; fax: +55 (14) 3880 0573.

E-mail addresses: braga_ca@yahoo.com.br, braga_ca@ibb.unesp.br (C.P. Braga).

Carla Mariane Costa Pozzi a, Camila Pereira Braga a,⇑, José Cavalcante Souza Vieira a, Bruna Cavecci a,João Vitor de Queiroz a, Herbert de Souza Barbosa b, Marco Aurelio Zezzi Arruda b, Fabio Cesar Gozzo b,Pedro de Magalhães Padilha a

a Institute of Biosciences, São Paulo State University-UNESP, Botucatu, SP, Brazilb Institute of Chemistry, University of Campinas-UNICAMP, Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 October 2013Received in revised form 4 April 2014Accepted 8 June 2014Available online 17 June 2014

Keywords:Flame atomic absorption spectrometryHuman milkElectrospray ionization–tandem massspectrometryMetalloproteomicsSecretory IgATwo-dimensional electrophoresis

The presence of calcium, iron, and zinc bound to human milk secretory IgA (sIgA) was investigated. ThesIgA components were first separated by two-dimensional polyacrylamide gel electrophoresis and thenidentified by electrospray ionization-tandem mass spectrometry (ESI MS MS). The metal ions weredetected by flame atomic absorption spectrometry after acid mineralization of the spots. The resultsshowed eight protein spots corresponding to the IgA heavy chain constant region. Another spot was iden-tified as the transmembrane secretory component. Calcium was bound to both the transmembrane com-ponent and the heavy chain constant region, while zinc was bound to the heavy chain constant regionand iron was not bound with the identified proteins. The association of a metal ion with a protein isimportant for a number of reasons, and therefore, the findings of the present study may lead to a betterunderstanding of the mechanisms of action and of additional roles that sIgA and its components play inhuman milk.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

There increased appreciation of feeding human milk in recentyears, because composition promote optimizing infant growthand healthy development (Ballard & Morrow, 2013 and Innis,2014). Breast milk provides all nutrients, vitamins, and mineralsthat newborns need for growth during the first six months of life,and no other fluids or food are necessary; therefore exclusivehuman milk feeding for the first 6 months of life, with continuedbreastfeeding for 1–2 years of life or longer, is recognized as thenormative standard for infant feeding (World Health Organization,2003 and Ballard & Morrow, 2013). Moreover, breast milk containsantibodies and many protective factors that assist in fighting dis-eases (WHO, 2009).

The most abundant proteins of human milk are casein,a-lactalbumin, lactoferrin, secretory IgA (sIgA), lysozyme, andserum albumin. The predominant antibody in breast milk is thesecretory immunoglobulin A (sIgA) (>90%) (Lönnerdal, 2010 and

Newburg, 2005). Each monomer of the 420-kDa dimeric immuno-globulin sIgA consists of two light chains, which are the same forall immunoglobulin classes, and a heavy chain known as the alphachain, which has a molecular mass of approximately 60 kDa. Theheavy chain consists of four domains: one variable and three con-stant domains. The two monomeric portions are held together bytwo polypeptides, a protein known as the ‘‘J chain’’ and the secre-tory component. The secretory component protects sIgA frombeing degraded by proteolytic enzymes, allowing its survival inthe gastrointestinal tract and enabling it to combat infectiousagents (Kerr, 1990; Lönnerdal, 2003; Rojas & Apodaca, 2002 andNewburg, 2005). When the child is breastfed, sIgA, which is resis-tant to digestion, accumulates in the intestine and prevents bind-ing and invasion by bacteria, viruses, parasites, and toxins in themucosal surfaces (Newburg & Walker, 2007).

Many proteins require a metal ion to perform their catalyticactivities, stabilize their tertiary or quaternary structures and/orpromote redox reactions (Mounicou, Szpunar, & Lobinski, 2009).In milk, two examples of proteins that utilize metals are serumalbumin, which contributes to the bioavailability of zinc(Blindauer et al., 2009; Lönnerdal, Hoffman, & Hurley, 1982 andFanali et al., 2012), and lactoferrin. When bound to iron, lactoferrinhelps fight pathogens by depriving them of an essential nutrient

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for their growth (Lönnerdal & Atkinson, 1989 and Permyakov,2009). Although sIgA is a widely studied protein, little attentionhas focused on its ability to bind metal ions or on the potential con-sequences of this binding. One exception involved a study byOdintsova, Zaksas, Buneva, and Nevinsky (2011) who determinedthat the catalytic activity of sIgA onto b-casein is greatly influencedby the presence of metal ions.

The functional and structural characterizations of these pro-teins at the genomic scale can be studied by metalloproteomics,a relatively new field of study that combines proteomic techniques,such as two-dimensional electrophoresis and mass spectrometry(MS), with techniques for metals analysis, such as atomic absorp-tion spectrometry, inductively coupled plasma (ICP MS)/induc-tively coupled plasma optical emission spectrometry (ICP OES),immobilized-metal affinity chromatography (IMAC), and toolsbased on synchrotron radiation (Haraguchi, 2004 and Shi &Chance, 2011). The combined use of these techniques can helpincrease the understanding of the biological and environmentalissues related to metal ions (Arruda & Azevedo, 2009).

In this context, the present study involves an investigation ofthe calcium, iron, and zinc associated with sIgA found in breastmilk using flame atomic absorption spectrometry (FAAS) followingprotein fractionation by two-dimensional electrophoresis (2D-PAGE) and identification by electrospray ionization-tandem massspectrometry (ESI MS MS).

2. Materials and methods

2.1. Materials

Ultrapure water (18.2 MX cm�1) was purified in an Elga PUR-ELAB Ultra Ionic system. The buffer solutions, acid mineralizationsolutions, and calcium, iron and zinc standards were preparedusing analytical-grade acetic (J.T. Baker) and phosphoric (Mallinck-rodt) acids and spectroscopic-grade nitric and hydrochloric acids(Merck). All of the organic solvents used (e.g., ethanol and metha-nol) were of analytical grade and were supplied by Merck. Thesolutions used during the electrophoretic separations and for theprotein standards were prepared with reagents of analytical purityfrom Amersham Biosciences. The protease inhibitor cocktail waspurchased from Roche. Coomassie Blue G-250 (J.T. Baker) andbovine serum albumin (Merck) were used for the determinationsof total protein. Standard calcium, iron and zinc solutions wereprepared by dilution of Merck Titrisol standard and lanthanum.All other reagents were of at least analytical reagent grade.

2.2. Sample collection and preparation

Milk samples were collected from five healthy mothers in thecity of Botucatu, São Paulo, Brazil on days 25, 41, 39, 40 and 21of lactation. The full procedure was approved by the ethics com-mittee of the Botucatu Medicine School. The five samples werecombined and then stored at �20 �C. At the time of the analysis,they were thawed and centrifuged at 4000g and 4 �C for 30 minto remove the fat layer.

2.3. Determination of protein concentration

The total protein concentration of the breast milk samples wasdetermined by the Biuret method (Doumas, Bayse, Borner, &Carter, 1981) using bovine serum albumin as the protein standard.The analytical calibration curves were built with concentrationsfrom 10 to 100 g/L from a stock solution of bovine serum albumin(100 g/L). A total of 50 lL of sample/standard and 2.5 mL of Biuretreagent was mixed and placed in a water bath at 32 �C for 10 min.

After 5 min at room temperature, the readings were performed at545 nm.

2.4. Two-dimensional electrophoresis

The defatted milk was diluted into a solubilization solution con-taining 7 mol L�1 urea, 2 mol L�1 thiourea, 2% (w/v) CHAPS, 0.5% (v/v) ampholytes at pH ranging from 4 to 7, 0.002% bromophenol blue(w/v), and a cocktail of protease inhibitors so that proteins wouldbe disaggregated and solubilized. A total of 250 lL of this solution(375 lg protein) was added to 13 cm strips containing precastpolyacrylamide gel with ampholytes immobilized at pH 4–7. Thesestrips were placed onto a focusing tray, where they remained for12 h at room temperature to be rehydrated with the proteinextract. After this period, the rehydrated strips were placed intothe isoelectric focusing unit Ettan IPGphor (GE Healthcare) forthe first-dimension separation of 2D electrophoresis, which wasperformed for a total of 18,000 Vh. Then, the strips were reducedfor 10 min with a solution containing 6 mol L�1 urea, 2% (w/v)SDS, 30% (v/v) glycerol, 50 mmol L�1 Tris–HCl, 0.002% (w/v)bromophenol blue, and 2% (w/v) DTT and alkylated for 10 minwith a similar solution, but DTT was replaced by 2.5% (w/v)iodoacetamide.

After equilibrating the strips, the second dimension of the elec-trophoretic process (SDS–PAGE) was performed. The strips wereplaced onto a 12.5% polyacrylamide gel that was previously pre-pared on a 180 � 160 � 1.5 mm glass slide. A piece of filter paperwas placed on the polyacrylamide gel next to the strip, onto whichwas applied 6 lL of a molecular mass standard containing theproteins b-phosphorylase (97.0 kDa), albumin (66.0 kDa), ovalbu-min (45.0 kDa), carbonic anhydrase (30.0 kDa), trypsin inhibitor(20.1 kDa), and a-lactalbumin (14.4 kDa). The strip and filter paperwere sealed with a hot solution of 0.5% (m/v) agarose to ensuretheir contact with the polyacrylamide gel. The second dimensionof the electrophoretic run was then performed in a 2D-PAGE elec-trophoresis unit, the Ettan Dalt (Amersham Biosciences, Uppsala,Sweden), in two stages: 7.5 mA/gel for 15 min and 15 mA/gel for4.5 h.

The proteins were revealed using a colloidal Coomassie stainreagent that consisted of a solution of 8% (m/v) ammonium sulfate,1.6% (v/v) phosphoric acid, 0.08% (m/v) Coomassie blue G-250, and25% (v/v) methanol. Before staining, the proteins were fixed in thegel for 60 min using a solution containing 10% (v/v) acetic acid and40% (v/v) ethanol. The dye remained in contact with the gel for72 h, after which it was removed in successive washes with deion-ized water. The resulting gels were then scanned using ImageScan-ner III (GE Healthcare), and the images were analyzed byImageMaster 2D Platinum 7.0 (GeneBio, Geneva, Switzerland).

2.5. In-gel digestion of protein spots and ESI MS MS analysis

The protein spots were removed from the polyacrylamide gel,and each one of them was placed into a well of a plate called theZip Plate (Millipore), which was where the tryptic digestion wasperformed. The In-Gel Digest-ZP Kit (Millipore) was used to digestthe proteins with trypsin and subsequently purify the obtainedpeptides.

Liquid chromatography coupled with mass spectrometry (LC–MS) analyses of the peptides present in the protein digests wereperformed on a Waters nanoAcquity UPLC chromatograph coupledto a Waters Synapt HDMS spectrometer that was equipped with anano-ESI source. Typically, 2–5 lL of the liquid sample wasinjected by the autosampler of the UPLC system and directed to aguard column (Waters Symmetry C18, 20 mm � 180 lm i.d., 5 lmparticles), where they were desalinated for 3 min at a flow of5.0 lL/min of 97:3 H2O/acetonitrile (MeCN) with 0.1% (v/v) formic

Table 1Linear gradient applied during protein elution.

Time (min) % A (H2O with 0.1%v/v formic acid)

% B (MeCN with 0.1%v/v formic acid)

0 97 35 85 1510 65 3522 40 6026 3 9728 97 330 97 3

Fig. 1. Experimental workflow.

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acid, before being transferred to the analytical column (WatersBEH130 C18, 100 mm � 100 lm i.d., 1.7-lm particles) and elutedat a flow rate of 1.0 mL/min, according to Table 1.

Peptide detection was performed online by the mass spectrom-eter, which was configured to operate in data-dependent acquisi-tion (DDA) mode in five sequential functions: one full-scan MSfunction (200–2000 m/z), three MS MS functions, and one functionof external standard calibration (lock mass). Additional MS param-eters included the capillary voltage at 3.0 kV, the cone voltage at30 V, source temperature at 100 �C, nano-ESI gas flow at0.5 L h�1, the trap and transfer cell collision energies at 6 and4 eV, respectively, and the detector at 1700 V. The full-scan MSand MS MS product ion spectra were obtained at a rate of 1 spec-trum s�1. Prior to analysis, the instrument was calibrated withphosphoric acid oligomers [0.05% (v/v) H3PO4 solution in 50:50H2O/MeCN] from 90 to 1960 m/z. Argon was used as the collisiongas at a pressure of 9.7 � 10�3 mbar.

The LC ESI MS MS runs were processed with the ProteinLynxGlobal Server v.2.2 software (Waters) and analyzed by databasesearch using the MASCOT v.2.2 system (Matrix Science). Theselected search parameters were as follows: trypsin digestion withup to 1 missing cleavage site, oxidation of methionine residues as avariable modification, and the mass tolerances of the peptides andfragments at ±0.5 Da. Searches were performed using the NCBInrdatabase.

2.6. Determination of metal ions in the protein spots

The calcium, iron, and zinc in the protein spots identified assIgA were determined by FAAS after mineralizing the samples ina microwave oven. The analyses were performed with spots fromtwo different electrophoretic runs, and gels were obtained in dupli-cate for each run.

The calcium, iron, and zinc determinations were performedwith a Shimadzu AA-6800 atomic absorption spectrometer thatwas equipped with background absorption correction with a deu-terium lamp and a self-reverse (SR) system. Hollow cathode lampsof calcium, iron and zinc (using the following wavelengths:422.7 nm, 248.3 nm and 213.0 nm, respectively) operating with acurrent of 10 mA and a spectral resolution of 0.5 nm were used.The analytical curves were prepared using Merck Titrisol standardsolutions, and the region of the gel where no protein spotsappeared was used for the analytical blank. The analytical curvesfor calcium, iron and zinc were constructed in concentrationsranges of 0.10–1.00 mg L�1

, 0.20–1.20 mg L�1 and 0.05–0.80 mg L�1,respectively. The analytical curves obtained for calcium, iron andzinc corresponded to the equations:

CðCaÞ ¼ Ab:þ 0:0180=0:0213 ð1Þ

CðFeÞ ¼ Ab:þ 0:00074=0:056 ð2Þ

CðZnÞ ¼ Ab:� 0:0045=0:2176 ð3Þ

where C(Ca), C(Fe) and C(Zn) is the concentration of calcium, iron andzinc in mg L�1 and Ab. is the absorbance signal.

The methodology metal ions determination was validated usingthe spiked acid extract of the analytical blank that were spiked with100 mg of reference material (Bovine Muscle Powder, RM 8414 –National Research Council Canada), containing 145 ± 20 mg kg�1 ofcalcium, 71.2 ± 9.2 mg kg�1 of iron and 142 ± 14 mg kg�1 of zinc.The volume of the acid extract obtained for reference materialwas adjusted to 5 mL with ultrapure water. This solution wasdiluted five hundred times to perform the calculation of the limitof detection (LOD) and limits of quantification (LOQ) of the methodof analysis.

The experimental procedures are summarized in Fig. 1.

3. Results and discussion

3.1. SIgA fractionation and identification

To identify and analyze sIgA, the proteins in breast milkunderwent a two-dimensional fractionation process. They werefirst separated according to their isoelectric point (pI) in 13-cmstrips over a pH range of 4–7, followed by the second dimensionof the electrophoresis process (SDS–PAGE) in which the proteinswere separated according to their molecular mass in 12.5% poly-acrylamide gels. The proteomic profile of breast milk obtainedfrom a pool of five donors within the pH range of 4–7 is shownin Fig. 2. Nine spots were identified as sIgA components using ESIMS MS (Table 2). Spot 1 was identified as the transmembranesecretory component, also known as poly-Ig-receptor. It exhibiteda pI of 5.76 and a molecular mass of 99,945 Da. The other eightspots, located within a pH range of 5.44–6.50 and each with amolecular mass of approximately 78,000 Da, were identified asthe alpha-1 heavy chain constant region.

Fig. 2. Human milk 2-D gel. The bottom image shows the spots corresponding tothe sIgA components, marked and numbered from 1 to 9.

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3.2. Validation of determination method of calcium, iron, and zinc byFAAS

The accuracy of the method was assessed using the RM 8414certified standard. The calcium, iron and zinc concentrations deter-mined for the RM 8414 were (n = 6): calcium – 142 ± 2.5 mg kg�1,

Table 2Spots identified as components of sIgA.

Spot Protein Acce

1 Transmembrane secretory component AAB22 Ig alpha-1 chain C region P2073 Immunoglobulin alpha-1 heavy chain constant region AAC84 Immunoglobulin alpha-1 heavy chain constant region AAC85 Immunoglobulin alpha-1 heavy chain constant region AAC86 Immunoglobulin alpha-1 heavy chain constant region AAC87 Immunoglobulin alpha-1 heavy chain constant region AAC88 Immunoglobulin alpha-1 heavy chain constant region AAC89 Immunoglobulin alpha-1 heavy chain constant region AAC8

Table 3Determination of calcium, iron, and zinc associated with the sIgA components.

Spot Protein

1 Transmembrane secretory component2 Ig alpha-1 chain C region3 Immunoglobulin alpha-1 heavy chain constant region4 Immunoglobulin alpha-1 heavy chain constant region5 Immunoglobulin alpha-1 heavy chain constant region6 Immunoglobulin alpha-1 heavy chain constant region7 Immunoglobulin alpha-1 heavy chain constant region8 Immunoglobulin alpha-1 heavy chain constant region9 Immunoglobulin alpha-1 heavy chain constant region

LOQ (Limit of Quantification).

iron – 69.1 ± 1.2 mg kg�1and zinc – 139 ± 2.2 mg kg�1. Observedthat the concentration value determined is close to the certifiedvalue, considering that the RM 8414 has: 145 ± 20, 71.2 ± 9.2 and142 ± 14 mg kg�1 of calcium, iron and zinc, respectively. It is alsoobserved that the relative standard deviation obtained are lessthan two percent, which proves that the determination methodpresent great precision (repeatability and reproducibility). The lim-its of detection (LOD) and quantification (LOQ), calculated based onthe standard deviation of 20 readings of the standard solutionblank and on the slope of the analytical curve (LOD = 3r/slopeand LOQ = 10r/slope), were: 2.50 and 8.30 lg L�1; 2.90 and9.70 lg L�1 and 2.20 and 7.30 lg L�1 for calcium, iron and zinc,respectively (Alcantara et al., 2004). The LOD and LOQ of themethod, calculated considering the standard solution blankcontaining 100 mg of RM 8414 (diluted five hundred times)were (mg kg�1): calcium – LOD = 16 (which corresponds to0.0032 mg L�1), LOQ = 51 (which corresponds to 0.0106 mg L�1),iron – LOD = 17 (which corresponds to 0.0034 mg L�1), LOQ = 55(which corresponds to 0.0113) and zinc – LOD = 14 (which corre-sponds to 0.0028 mg L�1), LOQ = 48 (which corresponds to0.0093), respectively (Moraes et al., 2013). It can be observed thatthe concentrations values determined for protein spots were allhigher than the method’s LOQ (Table 3).

3.3. Calcium, iron, and zinc bound to the protein spots characterized byESI MS MS

Human milk is an excellent source of Ca and can supply moder-ate amounts of Zn and Fe, small quantities of Mg, and traceamounts of Mn and Cu (Luo et al., 2010). Calcium is associated withthe formation of bones on newborn process, it is also essential tothe integrity of membranes, blood clotting and muscle contraction(Bates & Prentice, 1994). Regarding the iron present in breast milkis in smaller quantities than other foods, but their absorption andbioavailability are high. The total iron concentration is highest inthe first days postpartum, decreasing during the first month of lac-tation and maintaining more stable in the following periods (WHO,2009). The bioavailability of zinc in breast milk is high and used

ss code Score Observed pI/MM (Da) Coverage (%)

0203 57 5.76/99945 458 64 5.44/77800 52528 145 5.60/77800 122528 175 5.87/78678 172528 158 6.00/78972 172528 108 6.16/73830 52528 97 6.26/80764 52528 82 6.34/80764 52528 58 6.50/80764 5

Ca (mg kg�1) Fe (mg kg�1) Zn (mg kg�1)

82 ± 1.5 61 ± 1.1 <LOQ<LOQ <LOQ <LOQ<LOQ <LOQ 79 ± 1.4<LOQ <LOQ <LOQ<LOQ <LOQ <LOQ54 ± 0.9 <LOQ <LOQ<LOQ <LOQ <LOQ<LOQ <LOQ <LOQ<LOQ <LOQ <LOQ

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quite efficiently by newborns. Although infant formulas containthree times more zinc than milk, children who are breastfedmaintain higher levels of zinc in plasma than those fed by infantformula (Shi and Chance, 2011).

A significant portion of the calcium is associated with the lipidfraction and the aqueous fraction is present in low molar masscompounds or associated with whey proteins. The iron has struc-tural and catalytic functions in many proteins and/or cell compart-ments, and lactoferrin, a serum protein that binds iron with highaffinity, is considered responsible for its high bioavailability. Ofall the minerals that bind to proteins, zinc is the most abundantand most are associated with a serum albumin and lactoferrin(Luo et al., 2010).

A number of peptides from milk proteins with mineral bindingabilities have been reported (Vegarud, Langsrud & Svenning, 2010).Studies in vitro, suggests the caseins and their genetic variants, aremost often reported as precursors of peptides containing bindingsites, phosphoseryl and carboxyl, for different minerals (Pereset al., 1997). Since the non-phosphorylated peptides, mineralsseem to connect through other binding sites on the whey proteinssuch as lactoferrin, lactalbumin and lactoglobulin than in casein(Cayot & Lorient, 1997). The role of peptides with mineral bindingobtained from the other milk proteins, for example immunoglobu-lins are currently unknown.

So far, most related to the binding of metal to milk proteinswere made in vitro ions, which does not allow what happens in realmilk, scale studies that study resulted in important information onprotein activity and their associated components.

After identifying the sIgA components in the gel, calcium, iron,and zinc were determined by FAAS after acid mineralization of theprotein spots. The results presented in Table 3 are those that couldbe confirmed from the spots of two different electrophoretic runs.

The sIgA that is transmitted from mother to child throughbreast milk is a defense mechanism of great importance for thenewborn. More than 90% of the antibodies present in the milkare sIgA (Newburg, 2005). At birth, the newborn is sIgA-deficient,and it takes nearly 30 days after delivery until these antibodiesare produced at sufficient levels. During this period, therefore,the amount of sIgA that breast milk provides is at its maximumlevel in terms of the percentage of total protein (Walker, 2010).The protection that breast milk confers through sIgA and alsothrough other components gives breastfed children a sixfoldgreater chance of survival during the early months of life thanthose who are not breastfed (WHO, 2009). Considering sIgA’s roleand importance, it is easy to understand why it is a widely studiedprotein and why its structure is well known. In the present study,two sIgA components were separated and identified by 2D-PAGEand ESI MS MS, respectively. The first, alpha-1 heavy chain con-stant region was found in a sequence of eight spots, with pI valuesranging from 5.44 to 6.50 and a molecular mass of approximately78.000 Da. The fact that eight spots were identified as the samecomponent highlights the high resolving power of the 2D-PAGE,which allows visualization of different isoforms of the same pro-tein (Beranova-Giorgianni, 2003). In the case of sIgA, the variousisoforms may result from different levels of glycosylation. SIgA ishighly glycosylated, and there are specific glycosylation sites inits heavy chain constant region (Kerr, 1990 and Froehlich et al.2010).

The other identified component was the transmembrane secre-tory component, which has a molecular mass of 100 kDa (Kerr,1990) and is a transmembrane protein that binds the dimeric IgAto the basolateral surfaces of epithelial cells. This protein complexcrosses the mammary epithelial cell towards the apical surfaceand, during transport or at the moment it reaches its destination,the transmembrane component is cleaved by one or more prote-ases. The cleaved component that remains associated with the

dimeric IgA is called the secretory component, and this IgA com-plex, when released in the milk, is called sIgA. The fact that sIgAis resistant to proteolysis during the digestion process is attributedto the secretory component (Rojas & Apodaca, 2002 and Newburg& Walker, 2007).

These two sIgA components are commonly found in breastmilk proteomic studies (Hettinga et al., 2011; Liao, Alvarado,Phinney, & Lönnerdal, 2011, Molinari, Casadio, Hartmann, Livk,& Bringans, 2012 and Picariello et al., 2012). In a 2D-PAGE gelof human colostrum and mature milk, Murakami, Lagarde, andYuki (1998) identified both the IgA heavy chain constant regionand the transmembrane secretory component at positions in thegel similar to those of the present study. In a separate study(Gao et al., 2012) that quantitatively compared human transi-tional milk and mature milk proteomes, the IgA heavy chainand the transmembrane secretory component were specificallyused as sIgA indicators.

We were able to detect calcium bound to both sIgA componentsand zinc bound to the heavy chain constant region using FAAS. Themain advantage of this metalloproteomic strategy is that proteinsare isolated directly from the native milk environment, whichallows a more accurate analysis of the protein/metal ions associa-tions. This is the first time that the binding of a metal ion to specificimmunoglobulin A components is reported.

There is little information available on the binding of immuno-globulins and their components to metal ions in the literature. TheRadulescu (1995) found two copies of the sequence Cys-X3-His,which can bind to a metal ion in the IgA heavy chain constantregion. In the present study, the determination of calcium boundto that specific region of the sIgA corroborates what Radulescu(1995) foresaw.

However studies were conducted by Radulescu (1995), whosought in the constant regions of immunoglobulins shortsequences found that contained histidine and cysteine residues(Mounicou et al., 2009), which are known ligands for metal ions.Zinc is generally complex with amino acids, peptides and nucleo-tides and has an affinity for thiol and hydrogen groups. Neverthe-less, in all zinc metalloenzymes studied to date, the bindinggeometry observed most often is a slightly distorted tetrahedralwith the metal ion coordinating three or four protein side chains(McCall, Huang, & Fierke, 2000); not been reported in the literatureas zinc bind to sIgA.

One reason the binding of a protein in milk to a metal ion isimportant is because it facilitates the absorption of metals by thechild. This, however, does not seem to be the main function of sIgAwhen it binds to metals because it is not degraded in the gastroin-testinal tract (Davidson & Lönnerdal, 1987). Odintsova et al. (2011)identified a purpose for the metal binding during a study in whichthe specific hydrolysis of b-casein by sIgA in human milk was ana-lyzed. The authors found that the specific sIgA activity was medi-ated by metal ions, especially iron, followed by calcium, cobalt,and nickel.

The discovery of the binding of metal ions to specific compo-nents of sIgA can provide further information about the activitiesof these components. It is known that the free secretory compo-nent in breast milk performs activities that are independent ofits association with the other sIgA-producing components. Forexample, it can bind to the enterotoxigenic Escherichia coli, thepneumococcal surface protein A, and Clostridium difficile toxinA (Dallas & Rolfe, 1998). When in its free form, its 20 cysteineresidues, which are normally involved in disulfide bridgeformation, are also free, facilitating the binding of metal ions.It is possible that both these known activities of the secretorycomponent and the yet undiscovered activities are dependenton the binding of calcium, which could be the subject of furtherstudies.

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4. Conclusion

The determination of calcium, iron, and zinc bound to sIgA com-ponents can provide important information about the activity ofthe entire protein and its associated components. Very little isknown about the purpose of these bindings, but the results deter-mined from the present study represents an advance in the under-standing of these specific interactions and of the health andnutrition of breastfed newborns.

Acknowledgement

The authors gratefully acknowledge the financial support of SãoPaulo Research Foundation (Fundação de Amparo a Pesquisa doEstado de São Paulo, FAPESP, Processes 2010/51332-5 and 2011/03291-0) and the Government Funding Agency (Coordenadoria deAperfeiçoamento de Pessoal de Nível Superior - CAPES).

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