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Kiwifruit Actinidin: A Proper New Collagenase
for Isolation of Cells from Different Tissues
Ali Mostafaie & Ali Bidmeshkipour & Zeinab Shirvani &
Kamran Mansouri & Maryam Chalabi
Received: 30 April 2007 / Accepted: 21 November 2007 /
Published online: 3 January 2008
#
Humana Press Inc. 2008
Abstract Actinidin is a cysteine protease abundant in Kiwifruit. This enzyme is known as
a meat-tenderizing protease. In this project, actinidin was purified from kiwifruit by salt
precipitation and ion exchange chromatography. Collagenolytic effect of the purified
enzyme was tested in four different buffer systems. Thereafter, the enzyme was used for
isolation and culture of cells from three different tissues: endothelial cells from human
umbilical vein, hepatocytes from rat liver, and thymic epithelial cells from rat thymus. Our
results revealed that actinidin can hydrolyze collagen types I and II at neutral and alkaline
buffers. Furthermore, actinidin compared with type II or IV collagenase isolated intact
human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with
viability more than 90%. These results address a novel and valuable collagenase, which can
be used efficiently for hydrolysis of collagen and isolation of different cell populations from
various solid tissues.
Keywords Actinidin
.
Collagenase
.
Hepatocytes
.
Kiwifruit
.
Thymic epithelial cells
.
Umbilical vein endothelial cells
Introduction
Isolation of cells from various tissues essentially relies on disintegration of extracellular
matrix, which consists of various fibrillar proteins, glycoproteins, and proteoglycans.
Collagen is the main component of extracellular matrix that has important roles in
maintaining the adhesion and growth of cells [1, 2]. Fibers of collagens consist of three
chains that are wounded into a triple helical structure. The fibers provide the major
Appl Biochem Biotechnol (2008) 144:123–131
DOI 10.1007/s12010-007-8106-y
A. Mostafaie (*)
:
K. Mansouri
:
M. Chalabi
Medical Biology Research Center, Kermanshah University of Medical Sciences, Sorkheh Ligeh,
P.O. Box 1568, Kermanshah, Iran
e-mail: amostafaie@kums.ac.ir
A. Bidmeshkipour
:
Z. Shirvani
Department of Biology, College of Sciences, Razi University, Kermanshah, Iran
biomechanical scaffold for cell attachment and anchorage of macromolecules, allowing the
shape and form of tissues to be defined and maintained [3]. Due to its structure, collagen is
resistant to the action of ordinary proteases. Collagenases are the only enzymes that able to
cleave peptide bonds in the triple-helical regions of collagens [4, 5]. Two types of
collagenases are known: (1) microbial such as Clostridium histolytica collagenase that
generally split each polypeptide chain of collagen at multiple sites and (2) tissue collagenases,
the other type, have been found in vertebrate tissues undergoing growth or remodeling. The
vertebrate collagenases are distinguished by their ability to dissolve collagens by making a
single scission across all three α chains at a specific sensitive site [6].
Actinidin (EC 3.4.22.14) is a thiol protease, first characterized by Arcus [7]. This
enzyme is the major protein in most Actinidia fruits [8]. Actinidin is known as a good meat-
tenderizing enzyme. However, there are few studies on the collagenolytic activity of this
protease [9–11]. These studies did not report the hydrolytic effect of actinidin on collagen;
however, Morimoto et al. [11] reported that atelocollagen (pepsin-hydrolyzed collagen)
proved to be a substrate for actinidin at acidic pH. In the present project, collagenase
activity of actinidin toward types I and II collagen has been surveyed in different buffer
conditions. Furthermore, this protease has been used successfully for isolation and culture
of three different cell types: endothelial cells from human umbilical vein (HUVEC),
hepatocytes from rat liver, and thymic epithelial cells (TEC) from rat thymus.
Materials and Methods
Purification of actinidin Actinidin was purified from kiwifruit (Hayward cultivar) as
described by Boland and Hardman [12]. Briefly, the enzyme fraction was precipitated from
kiwifruit extract by 60% saturation of ammonium sulfate. The precipitate was redissolved
in 50 m
M citrate buffer (pH 5.5) and dialyzed overnight against this buffer. The dialyzate
was loaded into a DEAE-Sepharose Fast Flow column (Pharmacia), which pre-equilibrated
with the same buffer. The adsorbed fractions were eluted with 0.0–1 M linear gradient of
sodium chloride in the buffer.
Protein and protease assay Protein concentration was estimated by the method of Bradford
[13] using bovine serum albumin as the standard. Protease activity was determined based
on the method of Anson [14] using casein as the substrate.
Collagenase activity of actinidin Stock solutions (3 mg/ml) of collagen type I from rat tail
(Roche Applied Sci.) and type II from chicken external cartilage (Sigma Chemical Co.)
were prepared in distilled water adjusted to pH 3 with acetic acid. Solutions of collagen
type I or II with final concentration of 1 mg/ml were prepared from each of the stock
solutions in 20 mM acetate (pH 4), 20 mM citrate (pH 5.5), 20 mM phosphate (pH 7), and
20 mM Tris–HCl (pH 8.5) buffers. About 10 µl of actinidin (1 mg/ml) was added to 990 µl
of the collagen substrate solution in each buffer system. The reaction mixtures were
incubated for 1 or 2 h at 37 °C. Thereafter, the enzyme activity rapidly arrested by addition
of 250 µl of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer, and the tubes were incubated for 10 min in boiling water. Protein profile of
the all samples was analyzed by SDS-PAGE in 10% slab gels according to the method of
Laemmli [15]. After electrophoresis, the protein bands were stained with Coomassie
brilliant blue R-350 (Pharmacia) and analyzed by densitometry (Helena) in 600 nm to
determine the ratios of protein bands.
124 Appl Biochem Biotechnol (2008) 144:123–131
Isolation of HUVEC HUVEC were isolated and cultured using a modified standard
procedure [16]. Briefly, fresh umbilical cords were washed with sterile phosphate-buffered
saline (PBS). The umbilical vein was cannulated and thoroughly rinsed with sterile PBS
until the vessel is slightly distended to clear excess blood. After clamping the other
extremity, the vein was filled with 5–10 ml of 1, 2, 4, 8, or 16 mg/ml of actinidin or similar
concentrations of type II or IV collagenase (Sigma Chemical Co.). The cords were
incubated at 37 °C for 10, 20, 30, 40, 50, or 60 min to digest selectively the single layer of
endothelial cells. Thereafter, the vein was perfused with MCDB131 medium containing
20% fetal calf serum (FCS), and the released cells were collected by centrifugation (150×g
for 5 min). The pellet was resuspended in MCDB131 containing FCS, penicillin (100 IU/ml),
streptomycin (100 μg/ml), and endothelial cell growth factor (20 μg/ml). At confluence, the
endothelial cells were detached from the culture flasks using a solution of trypsin-EDTA and
passaged. The cells were then plated on culture plates or flasks precoated with gelatin.
Identification of endothelial cells HUVEC were identified by their non-overlapping
cobblestone morphology and immunostaining (Immunoperoxidase) with an antibody vs
factor-VIII-related antigen (Von Willbrond factor) and a secondary antibody (HRP-
conjugated) in representative plates.
Isolation of rat hepatocytes Hepatocytes were isolated from rat liver using the two-step
perfusion technique as described by Wang et al. [17] with some modifications. Wistar rats
(150–200 g from Razi Institute, Iran) were anesthetized with chlorophorm; then, heparin
was injected (200 IU per 100 g of body weight) into the femoral vein. The abdomen was
opened up to the sternum, and the vena cava closed with curved tweezers. The small
intestine was pushed towards the left side and the liver upward to cannulate the portal vein
(with 23-gauge needle). The cannula tightly was fixed with curved tweezers, and liver was
perfused with calcium and sulfate-free Krebs–Henseleit buffer at 10 ml/min for 15 min.
Few minutes after starting perfusion, one brunch of the portal vein was cut, and then liver
was perfused with 0.1, 0.2, 0.4, 0.8, or 1 mg/ml actinidin solution at 7 ml/min for 10, 15,
20, 25, or 30 min. The liver was taken out from the animal and placed within a Petri dish
together with the remaining actinidin solution for 20 or 30 min with gentle shaking for
further digestion. All the perfusion and digestion steps were kept at 37–38 °C. The digested
tissue was filtered through sterilized gauze and centrifuged for 3 min at 150×g. The pellet
was washed two further times via centrifugation steps. The final cell pellet was resuspended
in William’s E culture medium containing 10% FCS, 2.5 µl/ml amphotericine B, and 50 µg/ml
gentamycine and seeded in culture flasks precoated with collagen type I. The medium was
changed daily, and morphology of the cells was observed under microscope. The albumin
synthesis of hepatocytes was assessed using SDS-PAGE.
Isolation of rat TEC TEC from rat thymus were isolated as describe d by Ropke [18] with some
modifications. The rats were injected with dexamethasone (1 µl/g weight) and anesthetized
with chlorophorm 72 h after injection. The thy mus was taken out and washed with PBS
buffer. Th e gl and wa s minced into small pieces and suspended in the PBS containing 1, 2, 4,
8, or 16 mg/ml actinidin for 1, 2, 3, or 4 h at 37 °C wi th gentle shaking. All released cells we re
harvested at 15 0×g for 5 min and washed two times with PBS. The cell pellet was
resuspended in W illiam’s E culture medium containing 20% FCS, 2.5 µl/ml ampho tericine B,
and 50 µg/ml gentamycine. Th e cell suspensions were cultured in dishes precoated with
collagen type I at 37 °C. After severa l hours of incubation, the adherent cells were generously
washed with the culture medium three times and maintained in the same medium.
Appl Biochem Biotechnol (2008) 144:123–131 125
Viability Assessment
Isolated cells were washed with PBS and resuspended in this buffer. About 50 µl of trypan
blue solution was mixed with 100 µl of the cell suspension and allowed to stand for 2 min
at room temperature. The mixture then analyzed for cell viability according to the related
formula.
Results
Collagenase activity of actinidin The hydrolysis of types I and II collagen by actinidin
under different pH conditions was monitored by SDS-PAGE. Figure 1 shows the SDS-
PAGE pattern of collagen type I after incubation for 1 h with actinidin in acetate (pH 4),
citrate (pH 5.5), phosphate (pH 7) or Tris–HCl (pH 8.5) buffer system. As the figure
indicates, the protein bands of collagen type I completely disappeared after 1 h of
hydrolysis in Tris–HCl (pH 8.5) and phosphate (pH 7) buffer systems (lanes 8 and 6 vs 7
and 5 as their controls, respectively). In citrate buffer (pH 5.5), actinidin digested more than
half of the collagen type I (lane 4), but in acetate buffer (pH 4), the enzyme did not
hydrolyze this substrate (lane 2).
In comparison to collagen type I, actinidin hydrolyzed most of the collagen type II after
2 h incubation at Tris–HCl (pH 8.5) and phosphate (pH 7) buffer systems but did not
hydrolyze this protein in acetate (pH 4) or citrate (pH 5.5) buffer systems.
Fig. 1 SDS-PAGE of collagen
type I hydrolyzed by actinidin at
pH 4 (lane 2), pH 5.5 (lane 4),
pH 7 (lane 6), and pH 8.5 (lane
8). Lanes 1, 3, 5, and 7 are
controls, respectively. The gel
stained with Coomassie brilliant
blue
126 Appl Biochem Biotechnol (2008) 144:123–131
Isolation of HUVEC To find proper concentration of actinidin and necessary time
selectively to isolate the single layer of endothelial cells, different doses of actinidin (1 to
16 mg/ml) and different incubation times (from 10 to 60 min) were tested. The results
indicated that actinidin in concentration of 2–4 mg/ml for 20–40 min selectively isolates
HUVEC with minimal contamination from other cell populations (Fig. 2a). The viability of
separated cells was estimated more than 95% in these situations. The separated cells
showed morphologic and immunohistologic characters of HUVEC (Fig. 2b).
Isolation of rat hepatocyte Perfusion of 0.4 mg/ml actinidin solution at flow rate of 7 ml/min
for 15–20 min and an additional treatment for 20–30 min in Petri dish was isolated 2–3×10
7
hepatocytes from each liver with viability of 92–95%. Phase-contrast microscopy showed
that isolated hepatocytes were translucent and spherical in shape (Fig. 3a). After isolation,
the majority of intact hepatocytes adhered to each other, and the cells reconstructed their
cellular polarity after 48 h presenting a typical polygonal morphology and many with
binuclei (Fig. 3b).
Isolation of rat TEC Rat TEC was properly isolated after digestion of thymus in 4 mg/ml
actinidin for 4 h at 37 °C. The isolated cells were adhered to collagen precoated dishes after
washing. After 24 h of culture, the adherent cells were flattened and showed polygonal
morphology with small nuclei (Fig. 4a,b). The viability of the cells as judged by the trypan
blue test was estimated to be 90–95% in all isolations.
Discussion
Actinidin is a thiol protease abundant in kiwifruit [8]. This enzyme shows considerable
structural and functional similarities with other plant thiol proteases such as papain [19–21].
Actinidin is known as a meat-tenderizing protease [22]. Therefore, this suggests that it may
hydrolyze collagen. Although the effect of actinidin on synthetic or natural substrats has
been addressed in several studies [10, 23, 24], there are few reports on the collagenolytic
activity of this protease [9–11]. In the most recent study, Morimoto et al. [11] concluded
that actinidin has no collagenase activity, but atelocollagen (pepsin-hydrolyzed collagen)
proved to be a substrate for this enzyme at acidic pH.
Fig. 2 a Morphology of HUVEC 48 h and (b) 5 days after isolation (invert microscope ×400)
Appl Biochem Biotechnol (2008) 144:123–131 127
In the present project, we examined the hydrolytic effect of actinidin toward types I and
II collagens at four different buffers with pH 4, 5.5, 7, and 8.5. Our results revealed that
actinidin can digest the two types of collagens at pH 7 and 8.5 but that it had no
considerable effect on these substrates at acidic buffers particularly at acetate buffer with
pH 4. Furthermore, actinidin digested more efficiently type I collagen than type II, so that
the protein bands of collagen type I completely disappeared even after 30 min of hydrolysis
in Tris–HCl (pH 8.5) or phosphate (pH 7) buffers as assessed by SDS-PAGE (Fig. 1) and
protein assay methods. The pattern of SDS-PAGE indicates that actinidin could cleave
collagens at multiple sits. These results suggested that the mode of action of actinidin is
similar to bacterial collagenases. Type I collagen is the major structural constituent of most
connective tissues, except for cartilage, where homotrimeric type II collagen is prevalent
[25, 26]. Furthermore, types I and II collagen fibrils have been shown to have binding sites
for other types of collagens and proteoglycans [27, 28]. Therefore, tissue dissociation and
cell isolation is achieved by disintegration of these major types of collagens. The interstitial
collagenases are a group of endopeptidases having the ability to cleave the helical region of
native collagen fibers [29]. The definition excludes proteases capable of hydrolyzing only
the non-triple-helical telopeptide portions of collagens and enzymes capable of degrading
the triple-helical domains of collagens only in solution or extremes of pH.
Fig. 4 a Morphology of TEC after isolation (invert microscope ×100) and b 48 h after isolation
(Papanicolaou stained)
Fig. 3 a Morphology of hepatocytes after isolation (invert microscope ×400) and b 48 h after isolation
(Papanicolaou stained)
128 Appl Biochem Biotechnol (2008) 144:123–131
According to the meat-tenderizing effect of actinidin and collagenolytic properties of this
protease, which has been revealed in this study, we speculated that actinidin can be used for
isolation of different cells from various tissues. Hence, in this project, actinidin has been
used instead of collagenase to isolate HUVEC, hepatocytes from rat liver, and TEC from rat
thymus. The effect of actinidin on umbilical vein in different conditions showed that
optimal situation for selective isolation of HUVEC is 4 mg/ml actinidin for 20 min. In this
condition, the separated cells had viability of more than 95% and contained minimal
contamination from other cell populations as assessed by morphologic and immunohisto-
logic characters. These results were comparable with results of isolation of HUVEC by
collagenases in this study and other similar studies that successfully cultured endothelial
cells by collagenases treatment of large and small-sized vessels, including human umbilical
vein [16], human iliac vessels [30], bovine vena cava [31], rabbit pulmonary artery [32],
and heart vessels [33–35]. Endothelial cells form a single-cell layer that lines the inner
surface of all blood vessels. Much interest has been generated in isolation and culture of
endothelial cells due to their potential involvement in vascular disease, the repair of blood
vessels, and angiogenesis in cancer [36, 37].
Extracellular matrix of liver contains collagen types I, III, IV, V, and VI and various
glycoproteins [38]. For disintegration of collagenouse fine meshwork and isolation of
hepatocytes from rat liver, we used actinidin from 0.1 to 1 mg/ml in different situations. The
results indicated that perfusion of 0.4 mg/ml actinidin into the portal vein at 7 ml/min for
15 min and treatment of the taken-out liver for an additional time (15–20 min) in the enzyme
solution could isolate 2–3×10
7
intact hepatocytes from each liver . The isolated cells had
viability of 92–92% in all isolations and survived for at least 3 days in the culture flasks without
any growth factor. These results revealed that the use of actinidin in the basic two-step perfusion
procedure is a good choice for isolation of hepatocytes from rat and may be other animals. The
results were comparable with the results obtained herein and from other studies to used
collagenaeses in two-step perfusion method for isolation of hepatocytes from various rodents
[17, 39, 40] and human [41, 42] livers. Isolation and primary culture of hepatocytes is an in
vitro model widely used to investigate various aspects of liver physiology and pathology [43].
Thymus is a three-dimensional network of distinct cell types and extracellular matrix
elements [44]. These elements includes collagen types I and IV, laminin, fibronectin, and
various ligands of adhesion molecules. Unlike liver, disintegration of thymus extracellular
matrix needs harsh conditions using generally more than one type of hydrolytic enzymes.
So far, a mixture of three types of bacterial protease (Liberase) and DNase [45]or
collagenase and dispase [46, 47] has been used for disintegration of this tissue to isolate
TEC. In this study, TEC sufficiently were isolated after digestion of minced thymus in
4 mg/ml actinidin for 4 h. The isolated TEC had viability of more than 90% in all isolations
and showed normal morphology in culture. Isolation and culture of TEC provide a valuable
tool to study the role of these critical cells in thymus function [48].
Together, the present study clearly demonstrated collagenolytic activity of actinidin on
types I and II collagens, and used this protease for the isolation of HUVEC, hepatocytes,
and TEC for the firs time. According to the collagenolytic activity of actinidin, this protease
has a potential for isolation of different cell populations from various solid tissues.
Furthermore, acinidin can be isolated and purified from kiwifruit in large scale by a simple
method, without risk of infections, compared to bacterial or tissue collagenases. These
properties collectively address a novel and suitable collagenase for many applications in
cell isolation and in medical and biological sciences. However, the precise mode of action
of actinidin on collagen types I and II and hydrolytic effect of this protease on the other
glycoproteins of extracellular matrix need more quantitative investigations.
Appl Biochem Biotechnol (2008) 144:123–131 129
Acknowledgment We thank the staff of Hazrat Masomeh Hospital (Kermanshah, Iran) for collecting the
umbilical cords, Dr R. Ghorbani for morphological evaluation of the isolated cell, and Dr Z. Rahimi for the
revision of this article.
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