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36 I. DOLENI PEHAR et al.: Detection of cathepsin D in ewe milk, Mljekarstvo 63(1), 36-41 (2013)

*Corresponding author/Dopisni autor: Phone/Tel.: +385 1 2393 879; E-mail: skalit@agr.hr

Scientific note - znanstvena biljeka UDK: 637.112.2

Detection of cathepsin D in ewes milk

by Western Blotting method

Iva Doleni pehar1, Franjo Martinkovi2, Jasmina Havranek1,Albert Marinculi2, Milna Tudor Kalit1, Samir Kalit1*

1University of Zagreb, Faculty of Agriculture, Dairy Science Department,Svetoimunska 25, 10000 Zagreb, Croatia

2University of Zagreb, Veterinary faculty, Department of Parasitology and Invasive Disease,Heinzelova 55, 10000 Zagreb, Croatia

Received - Prispjelo: 19.11.2012.Accepted - Prihvaeno: 15.02.2013.

Summary

Milk contains about 70 indogene enzymes, while only twenty of them were investigated. Oneof the less explored is the milk enzyme cathepsin D, proteolytic enzyme located in the lysosomes,which are an integral part of the somatic cells whose number varies depending on the animals health.Unlike cows milk, in ewes milk the presence of cathepsin D was not determined, which could affectthe production of dairy products, especially cheese, which are traditionally produced in Croatia andMediterranean. This paper shows the presence of cathepsin D and its forms in ewes milk by modi-fied Western Blotting method. The analysis confirmed the presence of procathepsin D, mature andheavy chain of cathepsin D. Pseudocathepsin D and light chain of cathepsin D were not detected.

Key words: milk, cathepsin D, ewes milk, Western Blotting

Introduction

Milk contains a large number of indigenousenzymes which have different functions such asthe impact on dairy products quality and stabilityof processing procedures (Kelly and Fox, 2006).Among the indigenous enzymes, milk contains twomain proteinase systems, plasmin and lysosomalenzymes and other proteolytic enzymes (Fox andKelly, 2006). Plasmin as alkaline proteinase is themain proteolytic enzyme in raw milk (Andrews,1983; Kalit et al., 2002) and is associated to ca-sein micelles. One of the main lysosomal enzymes iscathepsin D (EC 3.4.23.5). It belongs to the groupof acid, aspartic endopeptidase and is present in lys-osomes of all mammalian cells. Cathepsin D (CatD)is originally considered as a house keeping enzymeinvolved in the clearance of unwanted proteins in thehuman organism (Ma rg aryan et al., 2010). Aminoacid sequence (primary structure) of procathepsin

D reminds on the amino acid sequences of other

aspartic proteases such as renin, pepsin and certainenzymes of fungi (Conner, 1992). According toavailable research, until now amino acid sequenceof procathepsin D has been identified only in somespecies of mammals such as human, bovine, mouse,rat, porcine and ovine which is shown in Table 1, anddashes in the ovine and porcine sequences indicatethat amino acid was not determined (Faust et al.,1985; Diedrich et al., 1990; Fujita et al., 1991;Larsen et al., 1993; Tyynela et al., 2000). Com-pared to human sequence, there is 70.45 % identitywith rat, 70.45 % with bovine, 69.23 % with ovineand 68.18 % with mouse procathepsin D (Be nes etal., 2002). CatD is involved in the metabolic deg-radation of intracellular proteins, activation anddegradation of polypeptide hormones and growthfactors, activation of enzymatic precursors, process-ing of enzyme activators and inhibitors, brain anti-gen processing and the regulation of programmedcell death (Vashishta et. al., 2009). In medical

research, CatD is known as a tumour marker and

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38 I. DOLENI PEHAR et al.: Detection of cathepsin D in ewe milk, Mljekarstvo 63(1), 36-41 (2013)

NuPage transfer buffer (Invitrogen). After blockingfor 30 min in 0.5 % PBS Tween 20, pH 7.5 (blockingbuffer), the membrane was incubated with custom-prepared human anti-goat cathepsin D monoclonalantibody (Neuromics Antibodies) (1:10000 in

0.05 % PBS Tween 20) for 1 hour at room tempera-ture. The membrane was then washed 3 times with0.05 % PBS Tween 20 and incubated for an hour anda half with anti-goat IgG-horse radish peroxidaseconjugate secondary antibody (Abcam) (1:10000in 0.05 % PBS Tween 20). After washing the mem-brane with 0.05 % PBS Tween 20, CatD-antibodycomplexes were detected using a combination of4-Chloro-1-naphthol and DAB solution.

Results and discussion

Cathepsin D (Cat D) and its forms were detect-ed in ewes milk using a modified protocol of WesternBlotting method due to the lack of appropriate anti-bodies. This study confirms that ewes milk containsthe enzyme cathepsin D and its forms: procathepsinD (two forms) 45 and 46 kDa, mature cathepsin D39 kDa and heavy chain 31 kDa, as shown in examplein Figure 1. Benes et al. (2002) detected procathep-sin in rat milk while Larsen et al. (1993, 1996) de-

scribed the bovine cathepsin D and its form in detail.

Although some authors believe that cathepsinD does not have a physiological role in milk, prob-lems arise when cheese-makers wants to producehigh quality dairy products manufactured frommilk containing an increased number of somatic

cells (Andrews, 1983; Fang and Sandholm, 1995;ODriscoll et al., 1999). Such milk is adverse forfurther processing primarily for the production ofcheese: curd retains more water, cheese is a soft,moist and elastic, resulting in a bitter taste and loweryield (Table 2). Such faults do not represent a prob-lem for large manufacturers (industry), where thereis a control of raw material (milk), but can be a prob-lem on small family farms. Traditionally, in Croatiain the area of Dalmatian hinterland, as well as inother low potential rural areas of Mediterranean ba-sin, sheep milk is used for production of high qualitycheeses (Bar bos a, 1990; Boyazo glu and Morand-Fehr, 2001; Ugarte et al., 2001; Matutinovi etal., 2007; Matutinovi et al., 2011), and any de-viation in quality of ewes milk can cause significantlosses. Further research should certainly go in thatdirection.

Western Blotting method was used to detect at-tendance of different forms of enzyme cathepsin Din ewes milk. The main problem was the absence

of adequate antibody which is the reason why for

Figure 1. Example of Western Blotting analysis of raw ewes milk.(M) Marker, (1) MCF7 Positive control, Human Whole Cell Lysate, (2, 3) different milk samples

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detection of cathepsin D antibodies that are rearedin the goat against a human cathepsin D were used.This antibody recognized human cathepsin D inewes milk. Secondary antibody, an anti-goat IgGpolyclonal antibody was linked to horse-radish per-oxidase in order to get better results. Addition ofsubstrate 4-Chloro-1-naphthol or DAB solution gaveno reaction so those two substrates were mixed andpoured over the membrane.

Although the Western Blotting method is high-ly sensitive and highly specific, enzyme-linked im-munosorbent assay (ELISA) is often used. Manyauthors in their research customized the method

of determination, so, ELISA was adjusted to addi-tion of haemoglobin (Larsen et al., 1993; 1996) or

synthetic heptapeptide (ODriscoll et al., 1999) assubstrate (Kelly et al., 2006). The lack of research

is certainly one of the reasons that there is still norapid, reliable and sensitive method for the determi-nation of enzyme cathepsin D and other proteases inmilk (ODriscoll et al., 1999).

Conclusion

Due to the fact that there are 50 unidentifiedenzymes in milk, determination of the enzymespresence in milk is crucial for investigation of thispart of dairy science. Large numbers of endogenousenzymes in milk have been identified but researches

on the presence of the enzyme in other milk typesare missing.

Product Enzymea Significance

Raw milk

LPO

XOR

Antimicrobial effect

Antimicrobial effect

Pasteurised milkPlasmin

AlP

Contributes to instability?

Index of processing

UHT milkPlasmin

SHOx

Contributes to gelation on storage?

Reduces cooked flavour

CreamLipase

LPO

Can cause rancidity

Indicator of heat treatment

Milk powdersLipase

Plasmin

Can cause rancidity

Can survive drying and remain active

Yoghurt

LPO

Plasmin

AcP

Inhibits post-acidification?

Affects gel structure and texture?

Dephosphorylates proteins and peptides?

Fresh cheese Plasmin Affects rennet coagulation of milk

Ripened cheese

General

Swiss

Acid

Plasmin

Lipase

Cathepsin D

Cathepsin D

Contributes to primary proteolysis

Contributes to lipolysis

May contribute to proteolysis due to inactivation of chymosin

May contribute to proteolysis due to low pH

Infant formulae LipasePlasmin

XOR

Can cause rancidityCan cause bitterness

Bactericidal effect

Rennet casein PlasminSurvives production and can act in products in which thisprotein is used

Table 2. Known or possible significance of indigenous milk enzymes for the manufacture and/or quality ofdairy products (Kelly and Fox, 2006)

aAbbreviations: AlP, alkaline phosphatase; AcP, acid phosphatase; LPO, lactoperoxidase; SHOx, sulphydryl oxidase; XOR, xanthineoxidoreductase

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