Isozymes of Lysozyme in Leukocytes and Egg White: Evidence for the Species-Specific Control of Egg-White Lysozyme Synthesis
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Proc. Nat. Acad. Sci. USA
Vol. 71, No. 5, pp. 1653-1657, May 1974
Isozymes of Lysozyme in Leukocytes and Egg White: Evidence
for the Species-Specific Control of Egg-White Lysozyme Synthesis
(leukocyte granules/bone marrow/hormones/antibacterial mechanisms/oviduct/muramidase)
ALEXANDER HINDENBURG*, JOHN SPITZNAGELt, AND NORMAN ARNHEIMt*
* Department of Biochemistry, State University of New York, Stony Brook, N.Y. 11790; and t Department of Bacteriology and
Immunology, School of Medicine, University of North Carolina, Chapel Hill, N.C. 27514
Communicated by Bentley Glass, February 15, 1974
ABSTRACT Two structurally distinct forms of egg- Canfield) and 3X crystalline chicken egg-white lysozyme
white lysozyme (EC 3.2.1.17) are known. The egg white of (Pentex) were the same as those used previously (6). In those
some species contains both of these forms, while the egg
white of other species appears to contain only one or the cases where lysozyme activity in a sample was to be measured
other of them. We have immunological and electrophoretic in the presence of rabbit antiserum, the antiserum was first
evidence that the chicken, which has only one lysozyme treated with Bentonite (0.5% final concentration) to remove
type in its egg white, contains both types in its poly- rabbit serum lysozyme.
morphonuclear leukocytes. Experiments on Embden
goose bone marrow showed that this tissue also contains Gel Diffusion Experiments. These studies were carried out
both Iysozymes, even though the egg white of this species in 1 % agar containing 0.15 M NaCl, 0.05 M Tris HCl (pH
contains only one of them. Our studies suggest that many
avian species have the genetic loci that code for both 7.45), and sodium azide as a preservative.
forms of lysozyme, but that a species-specific regulatory Lysoplate Enzyme Assays. The concentration of lysozyme
mechanism controls whether one or the other or both of
them are expressed during egg white production. The fact in the large granule fraction from chicken leukocytes was de-
that two distinct lysozymes are present in chicken leuko- termined by either the lysoplate method of Osserman and
cytes may be of significance to the antibacterial mecha- Lawlor (8) or a modification of this technique, where the
nism of these cells, especially in light of the fact that they lysoplates contained 1% Difco agar, 0.033 M sodium phos-
lack myeloperoxidase, an important leukocyte enzyme in
mammals. phate buffer (pH 6.2), 0.15 M sodium chloride, 0.5 mg/ml
of Micrococcus luteus (Worthington), and sodium azide as a
Two forms of lysozyme (EC 3.2.1.17) that differ radically preservative.
from each other in molecular weight (1, 2), amino-acid com-
position (1-4), amino-terminal amino-acid sequence (1, 2, 5), Antibody Absorption Experiments. Equal volumes of the
and immunological cross reactivity (6) have been found in preparation of large cytoplasmic granules, or in some cases
avian egg whites. This and other evidence (7) suggest that partially purified lysozyme, and either antibody against
these enzymes are the products of two distinct genetic loci. chicken egg-white lysozyme or antibody against Embden
One of these forms of lysozyme is typified by the enzyme goose egg-white lysozyme were mixed, incubated for one hour
found in chicken egg white ("chick-type" lysozyme), and at 230, and stored overnight at 4°. The supernatant was col-
the other form by the lysozyme found in the egg white of the lected after centrifugation at 1000 X g for 30 min.
Embden goose (3, 4) ("goose-type" lysozyme). Acrylamide Gel Electrophoresis. The "goose-type" and
The egg whites of some species appear to contain only a "chick-type" lysozymes were separated in 15% acrylamide
"chick-type" lysozyme (e.g., chicken and Peking duck), other gels without a stacking gel at pH 4.5 (9), with the buffers
species only a "goose-type" lysozyme (e.g., Embden goose); described by Brewer and Ashworth (10). The gels (in tubes
still others contain both types of lysozyme (black swan; refs. 13 cm X 0.5 cm) were run for 2.5 hr at a constant current
2 and 6; and Canada goose, ref. 7). of 5 mA per gel. After electrophoresis, they were fractionated
We present data that demonstrate that even though a into 0.2-ml samples in a Savant Auto-Gel Divider, with am-
species may have only one form of lysozyme in its egg white, monium acetate buffer (0.1 M acetate, pH 9.45), and sub-
it can contain both forms of the enzyme in other tissues. jected to further tests.
Our finding of multiple forms of lysozyme in chicken poly-
morphonuclear leukocyte (PMN) cytoplasmic granules and Bone Marrow was obtained from the leg bones of a goose of
in Embden goose bone marrow may eventually lead to new the Embden and Toulouse variety. The marrow was sonicated
information about the evolution of regulatory systems in in ammonium acetate buffer (0.1 M, pH 9.45), lyophilized,
vertebrates as well as on the antibacterial mechanisms of and stored at -20°.
lysosomes. A Large Cytoplasmic Granule Fraction from Chicken PMN
MATERIALS AND METHODS Was Prepared according to the method of Brune and Spitznagel
Antiserums and Proteins. Rabbit antiserums directed against (9). Briefly, 6.68 X 108 peritoneal exudate cells from chickens
purified Embden goose egg-white lysozyme (gift of Dr. R. (66% PMN, 17% lymphocytes, 9% monocytes, 8% eosino-
phils) were homogenized in 10 ml of 0.32 M sucrose with 3
Abbreviation: PMN, polymorphonuclear leukocyte. units of heparin per ml at 4°. Cells were homogenized with
t To whom reprint requests should be sent. a Kontes type D glass homogenizer fitted with a Teflon pestle
16531654 Biochemistry: Hindenburg et al. Proc. Nat. Acad. Sci. USA 71 (1974)
FIG. 1. Gel diffusion study of the immunological resemblance
between Embden goose egg-white lysozyme and chicken leukocyte
granule lysozymes. L, leukocyte preparation; AG, antibodies
directed against Embden goose egg-white lysozyme; and G,
purified Embden goose egg-white lysozyme.
until 50% were broken. Unbroken cells and large fragments
were removed by layering the preparation on 23% sucrose
and subjecting it to centrifugation for 15 min at 180 X g.
The granule-rich, post-nuclear supernatant upper layer was
then removed and subjected to gradient centrifugation. The
sucrose gradient consisted of a 30-ml lower, linear gradient
portion [56% (w/w) sucrose at the bottom to 38% sucrose
at the top] overlaid by 10 ml of 28% sucrose, and on top of
this 10 ml of 18% sucrose. The entire gradient contained
heparin at a concentration of 3 units/ml. A total of 3.9 ml
of the post-nuclear supernatant fluid (equivalent to 2.6 X
108 cells) was layered on top of the 13% sucrose and centri-
fuged under the following conditions: fotw2dt = 8 X 109
sec1 with Rma = 15.3 cm and Rmin = 6.4 cm. After cen-
trifugation the material was collected by upward displace- FIG. 2. Lysoplate analysis of chicken leukocyte granule
ment with 60% sucrose. lysozymes after absorption with antibodies against lysozyme.
L + AC and L + AG symbolize the supernatant obtained,
RESULTS after centrifugation, from a mixture of the leukocyte fraction
(L) and antibody against chicken egg-white lysozyme (AC) or
Purification of Large Leukocyte Granules. The large elongated antibody against Embden goose egg-white lysozyme (AG),
granules of the PMN were, as described (9), in contiguous respectively.
fractions of the gradient with the sucrose concentrations
ranging from 48.6% to 51.7%. These fractions were pooled Antibodies prepared against purified Embden goose egg-
and diluted to make the sucrose equal to 23%. The granules white lysozyme also reacted with the large granule fraction.
were collected by centrifugation, suspended in 1 ml of 0.32 M The granule enzyme, however, is immunologically related,
sucrose, and stored at -20°. but is not identical, to the Embden goose's egg-white lyso-
Immunological Identification of Granule-Associated Lyso- zyme, since the latter "spurs" over the former (Fig. 1). Since
zymes. These studies used antibodies directed against either the chicken and goose belong to taxonomically distinct orders
crystalline chicken egg-white lysozyme or purified Embden (11), differences in the structure and, therefore, in the im-
goose egg-white lysozyme. The antiserum directed against munological crossreactivity of these enzymes are not un-
the chicken enzyme does not react with Embden goose lyso- expected.
zyme; the antiserum directed against the Embden goose Additional studies also showed that the large granules from
enzyme does not react with chicken lysozyme (6). Thus, chicken leukocytes contain two distinct forms of lysozyme.
these two antiserums could be used to determine unambig- In these experiments the identification of the "goose-type"
uously whether the leukocyte granule fraction contained a and "chick-type" enzymes in the granule preparation was
"chick-type", an "Embden goose-type", or both types of based upon the inhibition of lysozyme activity observed after
lysozyme. incubation with antiserum (6). If the large granule prepara-
Antibodies prepared against crystalline chicken egg-white tion is mixed with excess antibody against chicken egg-white
lysozyme were found to react with extracts of the large granule lysozyme, we would expect this enzyme to be precipitated.
fraction in gel diffusion experiments. This granule enzyme The supernatant from this mixture, after centrifugation,
could not be distinguished from chicken egg-white lysozyme. should contain only the "goose-type" lysozyme. The presence
A precipitin line showing complete immunological identity of this lysozyme can be detected by enzymatic assay as mea-
formed between the two proteins. This result suggests, al- sured in lysoplates: agar plates that contain a turbid suspen-
though it does not prove, that this leukocyte granule enzyme sion of Micrococcus luteus. If the supernatant containing a
and egg-white lysozyme may be identical in amino-acid se- "goose-type" lysozyme is placed in a sample well in a lyso-
quence. plate, a clear halo will gradually develop around the well.Proc. Nat. Acad. Sci. USA 71 (1974) Isozymes of Lysozyme 1655
This is a consequence of the diffusion of the lysozym. mtg-
the agar and its hydrolytic action on the Micrococcus cells.
The identification of this enzymatic activity as being due to 15
a "goose-type" lysozyme rather than any residual "chick- 0
0
type" lysozyme is made by showing that the diffusion of this w
1o
lysozyme activity is inhibited by antibody against goose
egg-white lysozyme. Just such an experiment is shown in
Fig. 2-1. It can be seen that the supernatant from a mixture -5 1
of the large granule preparation and antibody against chicken
egg-white lysozyme (L + AC) has lysozyme activity and 5 10 15 20 25 30 35 40 45 78
FRACTON NO.
that this activity is inhibited in its diffusion by antibody
against goose egg-white lysozyme (AG) but not by antibody
20 B
against chicken egg-white lysozyme (AC). The result of a
similar experiment carried out with the supernatant from
a mixture of antibody against goose egg-white lysozyme and 15 ~~~~C
G
the large granule preparation (L + AG) is also shown in Fig.
2-2. Antibody against chicken egg-white lysozyme (AC), -J5
but not antibody against goose egg-white lysozyme (AG),
inhibited the diffusion of the enzymatic activity. These re-
sults confirm the presence of two distinct enzymes with lyso-
0 45 78
zyme activity in the large granules of chicken leukocytes. 5 10 15 20 25 30 35 40
These experiments furthermore rule out the possibility of a FRACTON NO.
single molecular species that has an immunological resem- FIG. 3. Electrophoretic separation of the "chick-type" and
blance to both chicken and Embden goose egg-white lyso- "goose-type" lysozymes present in chicken leukocyte granules (A)
or Embden goose bone marrow (B). Fractionation of -the gel
zymes, since either antiserum alone would in that case have was begun at the position in the gel to which the cationic tracking
been expected to precipitate all the lysozyme activity from dye migrated (tube no. 1). Peaks C and G contained the "chick-
the granule fraction. type" and "goose-type" lysozymes, respectively. The lysozyme
Quantitation of the Lysozyme Content in the Large Cytoplasmic concentration is expressed in ug/ml, as determined by lysoplate
Granules of Chicken PMN. The large granule fractions which assays using, wherever appropriate, purified chicken egg-white
lysozyme or Embden goose egg-white lysozyme as a standard.
had either the "chick-type" or the "goose-type" lysozyme
completely removed by absorption with antibody, were used
to estimate the amounts of the immunologically unrelated tion of lysozyme makes it seem unlikely that the enzyme we
forms of lysozyme in the granules. Known concentrations demonstrated in these large granules could merely have been
of chicken egg-white lysozyme diluted with an equal volume absorbed to them as a preparative artifact. Rather, the ob-
of a control rabbit serum were placed in wells in a lysoplate. servation tends to support the hypothesis, which awaits more
After incubation for 12 hr at 23°, the diameters of the halos rigorous proof, that the lysozymes detected were intrinsic
that formed around each well due to the diffusion of lysozyme to the large granules.
were measured and were found to be directly proportional Electrophoretic Separation of the Leukocyte Granule Lyso-
to the logarithm of the lysozyme concentration (8). By this zymes. Two distinct peaks of lysozyme activity were detected
procedure of standardization, the concentration of "chick- in acrylamide gels after electrophoresis of the granule frac-
type" lysozyme in the mixture of the large granule prepara- tion at pH 4.5 (Fig. 3A). The more basic peak (C) contained
tion and an equal volume of antibody against goose egg-white "chick-type " but not "goose-type " lysozyme, as judged
lysozyme was found to be 28 sg/ml. The concentration of the by antibody absorption experiments. The other peak (G)
"chick-type" enzyme in the undiluted granule fraction was contained only the "goose-type" enzyme. The recovery of
therefore 56 ,g/ml. Similar experiments were carried out on the "chick-type" and "goose-type" lysozymes from the gel
mixtures of antibody against chicken egg-white lysozyme was 90% and 70%, respectively.
and the granule fraction. In this case, known concentrations
of purified Embden goose egg-white lysozyme mixed with an Studies on Embden Goose Bone Marrow. A bone-marrow
equal volume of control rabbit serum were used as standards. extract from the Embden goose was also found to contain
The results showed that the large granule preparation from both a "chick-type" and a "goose-type" lysozyme. Fig. 4
chick PMN contains 44 /g/ml of "goose-type" lysozyme. shows the results of an antibody absorption experiment. The
This estimate could be subject to some error. The lysoplate supernatant from a mixture of the bone-marrow extract and
assay depends upon the rate of diffusion of the enzyme into antibody against goose egg-white lysozyme was found to
the Micrococcus-containing agar. Structural differences be- contain a lysozyme that was inhibited in its diffusion in lyso-
tween our control enzyme from Embden goose egg-white plates only by antibody against chicken egg-white lysozyme
and the chicken's "goose-type" lysozyme could result in (Fig 4-1). The supernatant from a mixture of the marrow
different diffusion rates by virtue of differences in the inter- extract and antibody against chicken egg-white lysozyme
action of each enzyme with the Micrococcus cells. Neverthe- also contained an enzyme inhibited in its diffusion only by
less, we estimate that since this granule preparation contained antibody against goose egg-white lysozyme (Fig. 4-2). Quan-
480 gg of protein per ml, the proportion of lysozyme in this titative estimates of the amount of each lysozyme present
preparation was (56 ,g/ml + 44 g/ml)/(O.480 mgofprotein in the marrow extract were also made. It contained 84 tg/ml
per ml) = 2084yg of lysozyme per mg of protein. This propor- of "chick-type" and 100ug/ml of "goose-type" lysozyme.1656 Biochemistry: Hindenburg et al. Proc. Nat. Acad. Sci. USA 71 (1974)
anisms during the evolution of these birds from their common
ancestors will require information on the molecular basis of
the control of egg-white lysozyme synthesis. Further insights
into the mechanism of control may come from studies on
the hormonal induction of egg-white protein synthesis in some
of these species. Experiments on chicken oviducts have al-
ready provided a good deal of information on the hormonal
regulation of the synthesis of egg-white proteins. Synthesis
of chicken egg-white ovalbumin, ovomucoid, conalbumin,
avidin, and lysozyme seems to be initiated in the oviduct
after hormonal stimulation (13-15). Ovalbumin and avidin
synthesis has been shown to be a consequence of an increased
synthesis of their respective messenger RNAs (16-18). It is
likely, therefore, that the species specificity of egg-white
lysozyme content is controlled by species-specific regulatory
mechanisms acting at the level of transcription or perhaps
translation. Other mechanisms that may affect the presence
of these lysozymes in egg white are less probable but cannot
be rigorously excluded until further experiments are carried
out.
Regardless of the exact molecular mechanisms by which the
egg-white lysozyme content of the different species was changed
during evolution, the question remains as to what the evolu-
tionary advantage was in altering the types of egg-white
FIG. 4. Lysoplate analysis of goose bone-marrow lysozymes lysozyme present in different species. One possibility is re-
after absorption with antibodies against lysozyme. M + AC and
M + AG symbolize the sqpernatant obtained, after centrifuga- lated to the function of these enzymes. It seems likely that
tion, from a mixture of the marrow extract (M) and antibody lysozyme has an antibacterial role in egg white, although
against chicken egg-white lysozyme (AO) and antibody against additional functions have been proposed for the enzyme in
Embden goose egg-white lysozyme (AG), respectively. other tissues (19, 20). Studies on the "goose-type" and "chick-
type" enzymes have shown that they are muramidases and
Two distinct peaks of lysozyme activity were also observed break down the bacterial cell-wall peptidoglycan by hydrolyz-
after acrylamide gel electrophoresis of the marrow extract ing the j3(1, 4) linkage between N-acetylmuramic acid and
(Fig. 3B). Peak C contained only the "chick-type" enzyme N-acetylglucosamine (21, 22). The two forms of lysozyme,
and peak G only the "goose-type" lysozyme, as judged by however, also differ in other catalytic properties (1, 3, 4,
antibody absorption experiments. The recovery of the "chick- 22-27). The "goose-type" enzyme has a 3- to 6-fold higher
type" and "goose-type" lysozymes from this gel was 67% specific activity and lacks the chitinase activity that is char-
and 60% respectively.
,
acteristic of the "chick-type" enzyme. It has also been sug-
gested that the "goose-type" enzyme may have a strong
DISCUSSION preference for those N-acetylmuramic-acid residues in the
The observation that the egg whites of the chicken and Peking bacterial cell wall that are substituted with a peptide (22).
duck appear to contain only a "chick-type" lysozyme, that It may be relevant that different species of bacteria vary
of the Embden goose only a "goose-type" lysozyme, and those in the degree of cross-linking and amino-acid composition
of the black swan and Canada goose both types of enzyme of the peptide portion of the peptidoglycan (28 29).
may be explained in either of two ways. The absence of a A second alternative excludes any direct effect of natural
particular lysozyme in egg white could reflect the absence selection on the presence or absence of each lysozyme type
of the structural gene for that enzyme in the genome of the because of the enzymatic properties of these proteins. It is
species. Alternatively, the structural genes for both forms possible that during evolution natural selection acted to affect
of lysozyme may exist in each species and the presence of the appearance of many different gene products in the egg
one or the other or both lysozymes in egg white would reflect whites of different species. The presence or absence of either
the action of a species-specific regulatory mechanism. form of lysozyme might therefore be a fortuitous consequence
Our data on granule-associated lysozymes of chicken PMN of such an event based, for example, upon a close linkage to
and Embden goose bone-marrow lysozymes clearly support other genetic loci.
the latter hypothesis. Additional confirmation comes from The presence of lysozyme activity in chicken PMN leuko-
data on the Peking duck. This species, which has "chick- cytes deserves comment from the point of view of the anti-
type" lysozymes in its egg white (summarized in ref. 12), bacterial function of these cells. Brune and Spitznagel (9)
appears to have a "goose-type" enzyme in other tissues (7). found two electrophoretically distinct forms of lysozyme in
The black swan (1, 2, 6) and Canada goose (7), on the other large cytoplasmic granules of these cells. We have identified
hand, have both types of lysozyme in their egg white. them as "chick-type" and "goose-type" enzymes. Brune
It would appear, therefore, that the presence of either or and Spitznagel (9) also observed that these cells lack the
both forms of lysozyme in the egg white of a particular species enzyme myeloperoxidase, which serves an important anti-
depends upon a species-specific regulatory mechanism. In- bacterial function in mammalian leukocytes. The absence
formation on the development of these species-specific mech- of this enzyme in chicken PMN leukocytes may be com-Proc. Nat. Acad. Sci. USA 71 (1974) Isozymes of Lysozyme 1657
pensated for by a broader spectrum of antibacterial -activities 9. Brune, K. & Spitznagel, J. (1973) J. Infec. Dis. 1-27, 84-94.
produced by the different catalytic properties of the "goose- 10. BeBrwer, J. M. & Ashworth, R. B. (1969) J. Chem. Educ. 46,
41-45.
type" and "chick-type" lysozymes. A recent report on human 11. Delacour, J. (1954) "The Waterfowl of the World," Country
PMN leukocytes indicates that, in addition to the one mur- Life, London, Vol. I.
amidase that has been found in those cells, a proteolytic en- 12. Prager, E. M. & Wilson, A. C. (1972) Biochem. Genet. 7,
zyme (elastase), which is able to digest the cell walls of some 269-272.
bacterial species, is also present (30). Further studies on avian 13. O'Malley, B. W., McGuire, W. L., Kohler, P. O. & Koren-
man, S. G. (1969) Recent Progr. Horm. Res. 25, 105-160.
and mammalian leukocytes will perhaps provide more in- 14. Palmiter, R. D. (1972) J. Biol. Chem. 247, 6450-6459.
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17. Means, A. R., Comstock, J. P., Rosenfeld, G. C. & O'Malley,
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3628 (J.S.). (1973) Nature 243, 331-335.
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