THE EFFECT OF TRYPSIN AND CHYMOTRYPSIN
ON THE ANTIBACTERIAL ACTIVITY OF
COMPLEMENT, ANTIBODIES, AND
LACTOFERRIN AND TRANSFERRIN IN BOVINE
COLOSTRUM
J.H. Brock, A. Piñeiro, F. Lampreave
To cite this version:
J.H. Brock, A. Piñeiro, F. Lampreave.
THE EFFECT OF TRYPSIN AND CHYMOTRYPSIN ON THE ANTIBACTERIAL ACTIVITY OF COMPLEMENT, ANTIBODIES, AND LACTOFERRIN AND TRANSFERRIN IN BOVINE COLOSTRUM. Annales de
Recherches Vétérinaires, INRA Editions, 1978, 9 (2), pp.287-294. <hal-00901004>
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THE EFFECT OF TRYPSIN AND CHYMOTRYPSIN
ON THE ANTIBACTERIAL ACTIVITY OF COMPLEMENT,
ANTIBODIES, AND LACTOFERRIN
AND TRANSFERRIN IN BOVINE COLOSTRUM
J.H. BROCK
Fundacíón F.
A. PIÑEIRO
F. LAMPREAVE
Cuenca Villoro, Instituto de Investigación Biopuimica y de
Gasco&ntilde; de Gotor, 4, Zaragoza-6, Spain.
Nutrición
&dquo;D. Juan Ca!los 1&dquo;.
Résumé
EFFET DE LA TRYPSINE ET DE LA CHYMOTRYPSINE SUR L’ACTIVITE ANTIBACTERIENNE DU COMPLEMENT, DES ANTICORPS, DE LA LACTOFERRINE ET DE LA TRANSFERRINE DU COLOSTRUM BOVIN. &horbar; L’effet de la trypsine et de la chymotrypsine a été
étudié sur les facteurs antibactériens du colostrum bovin. Le complément endogène du
Les IgM étaient attaquées par la
colostrum était très sensible à ces deux enzymes.
chymotrypsine, mais non par la trypsine. La trypsine attaquait lentement les IgGl, entrainant une perte d’activité biologique due au clivage à la fois des chaînes lourdes et légères.
Les IgG1 étaient seulement très légèrement attaqués par la chymotrypsine. La lactoferrine
et la transferrine non complexées au fer étaient toutes deux sensibles à la protéolyse,
mais les formes saturées en fer étaient plus résistantes et tendaient à former des fragments stables complexant le fer.
Introduction
It is well established that milk and colostrum contain a number of antimicrobial facSeveral of
tors (Reiter and Oram, 1967).
these have been shown to act in vitro against
potentially enteropathogenic organisms suggesting that they might be responsible for
the enhanced resistance of the suckled newborn to gastrointestinal infections and in the
case of ungulates, which absorb colostral
proteins into the circulation, to systemic
infection as well (Bullen et al., 1972 ; Reiter
and Brock, 1975 ; Reiter et at, 1975 ; Brock
et al., 1975). However, except in a few defined areas (for example the importance of
colostral immunoglobulin uptake by the gut
of the newborn calf or pig) the relevance
of the various antimicrobial activities de-
monstrated in vitro to protection in vivo is
ill defined.
One problem is that little is
known about the capacity of colostral antimicrobial factors to survive and operate in
the inimical environment of the gastrointestinal tract and, in particular, their ability to
either withstand or evade the activity of proIn vitro studies of the
teolytic enzymes.
effect of proteolysis on colostral components
have generally been limited to immunoglobulins, and have been directed towards
elucidation of their structure, whereas in vivo
assessment of proteolysis by investigating
the presence of colostral proteins or fragments in faeces gives little indication of the
situation in the upper gastrointestinal tract.
The work reported here is an attempt to
assess, by in vitro experiments, the relative
susceptibility of various colostral antimicro-
factors to pancreatic proteases, with
particular reference to the effect on their
These studies may
biological properties.
bial
enable attention to be focused on those
factors which are less affected by proteases
and which therefore have more chance of
playing a role in the protection of the newborn calf against gastroenteritis.
Materials and Methods
1. COLOSTRUM
Colostrum
was
obtained from the first mil-
king postpartum from cows known to be
free of intramammary infection. Whey was
obtained by precipitating the casein with
rennin followed by centrifugation and stored
frozen until
use.
2. TESTS FOR ANTIMICROBIAL
ACTIVITY
All experiments were based on the method
described by Reiter and Brock (1975).
2.1.
Activity
of
Endogenous Complement
Colostral whey, diluted 1:5 in 0.15 M NaCl,
with or without additions of proteases and/or
inhibitors, was tested for its effect upon an
inoculum of Escherichia Coli NCTC 8007 [serotype 0111 : K58(B4) : H2], as described inn
detail elsewhere (Brock et al., 1975).
2.2.
of
antibodies
Activity
Naturally Occurring
Colostral whey, or fractions thereof, were
tested for bactericidal activity against E. coli
NCTC 8007, as described fully elsewhere
(Brock et al., 1977 b). Normal bovine serum,
absorbed with the test strain of E. coli, was
used as a complement source at 1:10 dilution.
E. coli NCTC 8623 [serotype 0125:K70 (B15) :
H19], which was shown in preliminary experiments to be resistant to complement-mediated bactericidal activity but sensitive to
the lactoferrin bacteriostatic system.
3. PROTEOLYSIS
Trypsin (10,000 units/mg) was obtained
from Boehringer (Mannheim, W. Germany)
and a-chymotrypsin (9 - 11,000 units/mg) from
BDH (Poole, U.K.).
Except for experiments
with endogenous complement, where details
are given in the results, proteolysis was
carried out using 250 lA
g of active enzyme
per ml of colostral whey diluted 1:5 in 0.15M
NaCI.
However, it was necessary to take
account of the activity of colostral trypsin
inhibitor: in 1:5 diluted colostral whey this
was sufficient to inactivate 100 I
tg of trypsin,
but had only a negligible effect on chymotrypsin (Pineiro et al., 1975 ; Brock et al.,
1975) hence trypsin was normally added at
350 I
tg/ml to allow for this.
Proteolysis of purified IgGl, lactoferrin and
transferrin was carried out as described
In
elsewhere (Brock et al., 1976, 1977 a).
all experiments except those involving endogenous complement activity proteolysis was
terminated as required by adding’an excess
of either soybean trypsin inhibitor (Serva,
Heidelberg, W. Germany) or Trasylol (Bayer,
S.A., Barcelona, Spain).
4. GEL FILTRATION
AND ELECTROPHORESIS
Gel filtration of colostral whey was performed in gels of acrylamide-agarose AcA22,
and of digests of purified proteins in AcA44.
Electrophoretic analysis of digests was performed
using sodium dodecyl sulphate
(SDS)-polyacrylamide gels and on cellulose
acetate strips. Full details of these methods
have been given previously (Brock et al.,
1976, 1977 a, b).
5. PROTEIN
2.3. Bacteriostatic
PURIFICATION
Activity
The
lactoferrin - mediated
bacteriostatic
effect of bovine colostrum was activated by
adding NaHC0
)
°
3 (final concentration 0.1 °/
to colostral whey diluted 1:5 in 0.15M NaCl
according to a previously-described method
(Reiter et al., 1975). The test organism was
Bovine
isolated
lactoferrin and transferrin were
described previously (Reiter et
al., 1975 ; Brock et al., 1976) and colostral
immunoglobulins by the method of Butler
and Maxwell (1972).
Colostral trypsin inhibitor was prepared as described by Pineiro
et al. (1975).
as
6. IMMUNOGLOBULIN ASSAYS
of
bovine
(Pineiro
The concentrations of [gG, IgM and [gA
chromatographic fractions were assayed
by the method of Mancini et al. (1965).
in
et
chymotrypsin
by
this
inhibitor
al., 1978).
2. ANTIBODIES
Complete integrity of biological activity of
immunoglobulin molecule requires that
both the Fab regions, which contain the
antigen-binding sites, and the Fc region,
which mediates several non-specific functions including complement fixation remain
intact.
The complement-mediated bactericidal activity of naturally-occurring antiboan
Results
1. COMPLEMENT
The presence of complement has been
demonstrated in bovine colostrum by both
bactericidal and haemolytic activity (Brock
et al., 1975), but the level as measured by
these assays is much lower than in serum.
Bactericidal activity of complement in colostrum was exceedingly sensitive to trypsin,
since even 50 yg/ml of trypsin reduced the
bactericidal activity, despite the fact that
the colostrum contained sufficient trypsin
inhibitor to neutralise double that quantity
of trypsin (Fig. 1 a).
An excess of trypsin
caused complete loss of activity, which
could, however, be prevented if a large
excess of exogenous colostral trypsin inhibitor was added. Chymotrypsin was rather
less active in destroying bactericidal activity (Fig. 1 b) but its effect could not be
reversed by an excess of colostral trypsin
inhibitor, in accord with the weak inhibition
dies to E. coli has therefore been used to
test the effect of proteolysis on bovine colostral
immunoglobulins, since this activity
requires both the functions of the immunoglobulin molecule, and is also extremely
sensitive.
2.1.
Proteolysis
of Colostral
whey
Trypsin or chymotrypsin treatment of colostral whey caused a slow decrease in bactericidal activity measured in the presence of
exogenous complement, much slower than
the very rapid loss of activity occurring
when only endogenous complement was present to mediate the activity. Some inhibitory
activity was still present even after 24 h.
incubation (Fig. 2). With trypsin, the loss of
activity was initially rather rapid, but then
diminished, whereas with chymotrypsin the
loss of activity occurred more uniformly.
investigate which classes of antibodies
being affected, colostral whey was
chromatographed on a column of AcA22 acrylamide-agarose gel. Fractions were tested
for bactericidal activity and immunoglobulin
To
were
was found that most activity
associated with the IgG peak, and a
lesser amount with laM (Fig. 3).
When chymotrypsin-treated colostral whey
was similarly treated it was found that the
bactericidal activity associated with IgG
remained unchanged, but that associated
with IgM had been destroyed (Fig. 4). The
latter observation correlated with the fact
content, and it
was
that the elution position of the IgM peak
had been modified and corresponded to
a
molecular weight of about 620,000 as
compared with 950,000 for IgM in undigested
colostral whey.
Trypsin-treated colostral whey contained
intact [gm-associated bactericidal activity,
and the elution position of IgM was the same
The IgGas with undiges!ed whey (Fig. 5).
associated activity had, however, disappeared, although no obvious change in the
elution position of IgG was observed.
In summary, therefore, chymotrypsin preferentially destroyed IgM bactericidal activity, and trypsin the IgG activity, although
the latter event could not be correlated with
a
Neither
reduction in molecular size.
enzyme affected the elution position of colostral IgA, indicating that no gross fragmentation of this immunoglobulin had occurred.
Since no bactericidal activity was associated
with IgA it was, however, not possible to
say whether its biological activity had been
affected.
a) Purified colostral IgGl
was found to be
bactericidal in the presence of complement,
whereas IgG2 was inactive. This is in agreement with the poor complement fixing ability
of bovine IgG2 (Feinstein &
Hobart, 1969)
and confirms that the activity of the IgG
peak was due to igG1 and not to the small
amount of coeluting colostral IgG2.
b) Tryptic digestion of colostrum possessing
specific antiperoxidase activity (largely confined to the igG1 subclass) showed that loss
of bactericidal activity parallelled loss of
specific antibody activity.
c) Digests of purified normal and antiperoxidase igG1 analysed by gel-filtration in acrylamide-agarose AcA44 and by electrophoresis
in SDS-polyacrylamide gel showed that 73 °/o
of the material apparently remained undegraded after 6 h digestion, the remainder
being in the form of small peptides, and
with minimal amounts of Fab and Fc fragments. Nevertheless, antiperoxidase
reduced to only 55 °/o of the
was
activity
original
activity.
2.2. The Effect of
The
igG1
riments :
on
of the effect
elucidated by the
nature
was
Trypsin
IgG
of trypsin on
following expe-
d) SDS-polyacrylamide gel electrophoresis of
digests treated with 2-mercaptoethanol to
reduce disulphide bridges revealed that considerable cleavage of both light and heavy
chains had occurred.
From these results it appears that trypsin
acts on IgGl by causing chain cleavage in
the antigen binding region, rather than the
classical split to Fc and Fab fragments,
which occurred only to a very minor degree.
Similar experiments carried out with chymotrypsin-digested IgGl revealed that this
enzyme caused very little chain cleavage or
loss of antibody activity.
3. LACTOFERRIN AND TRANSFERRIN
The lactoferrin content of bovine colostrum
its iron
to permit the demonstration in vitro of an ironreversible bacteriostatic effect of colostral
whey on iron requiring organisms such as
E. coli, although this effect requires either
the addition of bicarbonate to or the elimination of citrate from the colostrum (Reiter
et al., 1975). Colostrum also contains variable amounts of serum transferrin, which may
also contribute to bacteriostasis (Schanba-
(1-4 mg/ml) is sufficiently high and
saturation (20-30 °/o) sufficiently low
cher and Smith,
1975). Specific antibodies
enhance the bacteriostatic
activity of lactoferrin (Bullen et al., 1972).
Trypsin caused a progressive loss in inhibitory activity and by 8 h all bacteriostatic
activity had been abolished (Fig. 6). Since
this loss of activity was much more rapid
than that observed for loss of specific antibody activity (see above) it seems likely that
trypsin was acting by destroying lactoferrin
(and/or transferrin) rather than any possible
are
reported
to
synergistic antibody activity.
Chymotrypsin
less active : little decrease in bacteriostatic activity was observable after 8 h incubation, and even at 24 h some inhibitory
activity remained.
Proteolysis by trypsin of purified bovine
lactoferrin and transferrin in both their iron
free (apo) and iron-saturated (Fe
-) states
2
revealed that the apo-forms were rapidly
digested : less than 10 °/o remained intact
after 3 h incubation, and both had been
almost completely degraded to small peptides by 24 h.
-lactoferrin was also
2
Fe
rapidly cleaved, but digestion only proceeded
as far as the production of large fragments,
which were largely resistant to further digestion. With Fe
-transferrin a similar process
2
was observed, but cleavage to large fragments was slower.
With both proteins the
absorption at 470 nm due to the coloured
Fe-protein complex was scarcely altered by
proteolysis, indicating that the large fragments so formed still retained iron-binding
capacity. This was confirmed by celluloseacetate electrophoresis of the digests, in
which it was observed that some of the
bands corresponding to fragments gave a
positive reaction with a specific stain for
iron. Two different fragments of transferrin
have been isolated and characterised, and
been shown to correspond to the N- and
C-terminal halves of the molecule, each
containing one iron-binding site (Brock and
Arzabe, 1976 ; Brock et al., 1978).
Attempts to isolate the lactoferrin fragments which are observed in SDS electrophoresis have, however, been unsuccessful,
since gel filtration under a variety of conditions (high
salt concentration, low pH,
6M urea) consistently yielded only small
amounts of free fragments, most material
eluting in a similar position to intact lactoferrin.
The fragments therefore remain
tightly but non-covalently bound to each
other, perhaps due to the same forces that
wass
caused lactoferrin to bind
to other proteins (Hekman,
Fragments capable
of
non-specifically
1971).
binding
iron
were
also formed transiently during tryptic digestion of apotransferrin and apolactoferrin, but
they were much less stable than the corresponding iron-saturated fragments formed by
digestion of Fe
-transferrin or lactoferrin.
2
Preliminary studies of the effect of chymotrypsin on purified lactoferrin and transferrin indicate that it acts in a qualitatively
similar manner to trypsin, but more slowly,
in agreement with its relatively weak effect
on
bacteriostasis.
Discussion
In the newborn calf, ability to produce
gastric acidity is relatively weak, and this,
combined with the buffering capacity of
colostrum, results in a temporary increase
in the pH of the stomach contents to 5 or
above (Mylrea, 1966).
Furthermore, antimicrobial factors will be contained largely in
the whey fraction rather than the casein clot,
and will therefore pass rapidly into the duodenum.
Consequently it is unlikely that
either gastric acidity or the acid proteases
pepsin and rennin will have much effect
on the antimicrobial factors, and the pancreatic enzymes are more likely to constitute
the major potential source of proteolytic
degradation. In a previous discussion of the
effect of these enzymes on antimicrobial factors in colostrum (Reiter and Brock, 1975) it
was evident that insufficient data were available for firm conclusions to be drawn.
Bactericidal activity of colostrum mediated
by naturally occurring antibodies and endogenous complement was exceedingly sensitive to proteases, especially trypsin, due to
destruction of complement. This is not surprising since the C3 component, required
for activation by both the classical and alternate pathways, is known to be very susceptible to proteolytic activity (Bokisch et al.,
1969). It therefore seems unlikely that bactericidal activity mediated by endogenous
complement in colostrum could be important as an antimicrobial system in the gut.
However, Day et al. (1969) demonstrated that
pig colostrum was rich in components C1
and C2 ant that the levels of these compo-
of suckled
The observation
bovine colostrum
(Brock et
augments the activation of complement by
the classical pathway (which reauires C1
and C2) but not by the alternate pathway
(which does not) suggests that bovine colostrum may also be rich in these components.
Thus a role of colostral complement in augmenting circulating complement levels in the
suckled newborn calf seems worth consinents
higher
were
in
the
sera
than in unsuckled
piglets.
al., 1975) that
dering.
IgGl is quantitatively by far the most
important immunoglobulin in bovine colostrum, and probably plays a crucial role in
providing circulating antibody in the suckled
calf.
However, there is
no reason to supalso play a role in
immunity, and indeed Newby and
Bourne (1976 a) have suggested that in the
bovine species igG1 fulfills some of the
functions normally performed in other species by the secretory IgA system, which is
relatively depressed in the bovine. Under
the conditions used in these experiments
destruction of tgG1 by trypsin was slow, and
by chymotrypsin minimal.
Thus this immunoglobulin should be fairly
resistant to proteolysis by pancreatic enzymes in the gut, especially as trypsin activity
is likely to be reduced by the presence of
colostral trypsin inhibitor (Pineiro et al.,
1978). IgM, on the other hand, would be
more susceptible to destruction, since chymotrypsin, to which it is more sensitive,
would be much less inhibited by the trypsin
inhibitor.
No conclusions can be drawn
from these experiments regarding the susceptibility of IgA and IgG, to proteolysis,
although we have confirmed (unpublished
results) the observations of Newby and
Bourne (1976 b) that bovine IgG, is more
An imsusceptible to proteolysis then
portant finding was that tryptic digestion of
IgG, causes loss of antibody activity in
molecules which in the absence of reduction of disulphide bridges appear to be
structurally intact. Thus caution should be
exercised in using gel filtration data as an
exclusive means of determining the degree
of proteolysis of colostral immunoglobulin
(Hardy, 1969 ; Kruse, 1973; Newby and
pose that
intestinal
it
may
not
l
igd,
The effect of
been studied.
Such data should be comfor specific antibody
chain cleavage.
Bourne, 1976 b).
plemented by
activity and/or
Trypsin was
tests
found to destroy the bacteriostatic effect of lactoferrin (and transferrin)
in bovine colostrum more rapidly than it
This is in
destroyed antibody activity.
accord with the finding that the susceptibility of lactoferrin and transferrin to proteolysis depends upon their iron saturation, the
unsaturated (a
o) protein being much more
D
susceptible. Similar results have been reported previously for human transferrin and
hen ovotransferrin (Azari and Feeney, 1958 ;
Williams, 1974). It seems difficult to reconcile these observations with the fact that
antimicrobial activity requires the apo-protein, and may perhaps indicate that the primary function of lactoferrin is not bacteriostasis.
Furthermore, lactoferrin, despite its locais secretory organs where it may
come into contact with proteases, appears
lisation
be if
anything more susceptible to prothan transferrin which is essentially
a serum protein.
Nevertheless, Bullen et al.
(1972) demonstrated that dosing suckling
guinea pigs with haematin increased their
susceptibility to experimental infection of
the gut by E. coli.
Further studies are
required to assess the significance of lactoferrin as an antimicrobial factor.
to
teolysis
It may be of significance that most of the
antimicrobial factors in colostrum investigated in this study were more susceptible
to trypsin than to chymotrypsin, IgM being
the only obvious exception.
The trypsin
inhibitor of bovine colostrum is much more
active against trypsin than against chymotrypsin (Pineiro et al., 1978) and hence its
selective action may serve to prevent degradation of these proteins without completely
inhibiting the digestive function of the pancreatic proteases.
It has been shown that
the duodenal contents of newborn colostrum-fed piglets are devoid of trypsin activity but possess chymotrypsin activity, and
also trypsin-inhibiting activity (Baintner, 1973).
Trypsin activity only appeared between 4 and
9 days after birth when trypsin inhibitor activity had disappeared.
Summary
trypsin and chymotrypsin on antibacterial
Endogenous complement in colostrum
factors
was
in
bovine colostrum has
sensitive to both
extremely
IgM was attacked by chymotrypsin but not by trypsin. Trypsin slowly attacked
igG1, causing loss of biological activity due to cleavage of both light and heavy chains.
IgGl was only very slightly attacked by chymotrypsin. Lactoferrin and transferrin in the
iron-free state were both susceptible to proteolysis, but the iron saturated forms were
more resistant and tended to give rise to stable iron-binding fragments.
enzymes.
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