VOl. 259, No . 18, Issue of September 25. PP. 11617-11625,1984 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 16. 1984 by The Amencan Society of Biological Chemists, Inc

Stomach Lysozymes of Ruminants 11. AMINO ACID SEQUENCE OF COWLYSOZYME OTHER LYSOZYMES*

2 AND IMMUNOLOGICAL COMPARISONS WITH (Received for publication, December 15, 1983)

Pierre Jollb$, Franqoise Schoentgen, and Jacqueline Jollb From the Laboratory of Proteins, University of Paris V, 45 rue des Saints-Peres, F-75270 Paris Cedex OS, France

Deborah E. Dobson$, Ellen M. Prager, and Allan C. Wilsonn From the Department of Biochemistry, University of California, Berkeley,California 94720

The complete sequence of 129 amino acids has been determined for one of three closely related lysozymes c purified from cow stomach mucosa. The sequence differs from those known for 17 other lysozymes c at 39-60 positions, at one of which there has been a deletion of 1 amino acid. The glutamate replacement at position 101 and the deletion of proline at position 102 eliminate the aspartyl-prolyl bond that is present between these positions in all other mammalian lysozymes c tested. This bond appears to be the most acidsensitive one in such lysozymes at physiological temperature. Of the 40 positions previously found to be invariant among lysozymes c, only one has undergone substitution in the cow lineage. This modest number of changes at novel positions is consistent with the inference, based on tree analysis and antigenic comparisons, that the tempo ofevolutionary change in the cow lysozyme lineage has not been radically different from that in other lysozyme c lineages. The mutations responsible for the distinctive catalytic properties and stability of cow lysozyme c could be a minor fraction of the total that have been fixed in thecow lineage.

The lysozyme c of ruminants provides an opportunity to examine both the driving force for protein evolution and the structural basis of altered protein function.As the result of a major regulatory change, this lysozyme appears to have lost its old function and acquired a new one. According to the hypothesis offered by Dobson et al. (1979, 1984), ruminant lysozyme c no longer works at or near neutral pH asa shield against bacterialinfection in many tissues and secretions (e.g. white blood cells, tears, egg white, and milk), as do the lysozymes c of many mammals and birds (Feeney and Allison, 1969; Osserman et al., 1974). Instead, in ruminants, this enzyme appears to be produced only by the stomach mucosa and tofunction exclusively as a major digestive enzyme (Dobson et al., 1979,1984) inthe presence of pepsin and ata lower -~

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by grants from the Centre National de la Recherche Scientifique (Equipe de Recherche No. 102) and the Institut National de la Santi et de la Recherche Medicale (Unit6 U-116). This is the 119th article on lysozyme from this author’s laboratory, the 118th being Berthou et al. (1983). I Present address, Dana-Farber Cancer Institute and Department of Pharmacology, Harvard Medical School, Boston, MA 02115. ll Supported by National Institutes of Health Grant GM-21509.

pH than is usually the case for the lysozymesof typical mammals and birds. An important step toward understanding the structural basis for functional change is to determine the amino acid sequence of a ruminant stomach lysozyme and to compare it with the sequences known for conventional lysozymes c from other animals. Seventeen complete sequences are known for non-ruminant lysozymes c (Jollbs et al., 1979a; Jung et al., 1980; Kondo et al., 1982; and references therein) and partial sequences are available for seven more (White, 1976; Jollbs et al., 1979a,1979b; Gavilanes et al., 1982; and references therein). We have, therefore, characterized and sequenced a lysozyme c from the stomach mucosa of a typical ruminant, the domestic cow. Furthermore, by conducting a tree analysis of the sequence data and making quantitative immunological comparisons of cow lysozyme c to otherlysozymes, it hasbeen possible to find out whether the rate of sequence evolution has been affected by the functional shift. EXPERIMENTALPROCEDURES’

Lysozymes and Tissues-The purified lysozymes: mammalian stomach extracts, and primate milks used in this study are described by Dobson et al. (1984). Enzymes and Reagents-Trypsin (EC 3.4.21.4) and carboxypeptidases A and B (EC 3.4.17.1, EC 3.4.17.2)were purchased from Worthington and Staphylococcus aureus V8 protease (EC 3.4.21.19) from Miles. Sephadex G-10, G-25 (fine), and G-50 (fine) were obtained from Pharmacia, and Bio-Gel P-60 from Bio-Rad. Cyanogen bromide was obtained from Merck. All other reagents (analytical grade) were purchased from Merck or Prolabo; those employed for the Sequencer were from Merck (Sequanal grade). For work other than sequencing, reagents were of standard analytical grade. Reduction, Alkylation, Citraconylation, and Enzymatic and Chemical Cleavage-Bovine stomach lysozyme 2 was reduced with 2-mercaptoethanol and alkylated with iodoacetamide according to Jollis et al. (1972). Reduced and alkylated lysozyme (20 mg) dissolved in 4 ml of 10 mM NaOH to which 4 ml of 50 mM N-ethylmorpholine were then added was citraconylated at pH 8.5 for 2 h at 20 “C (Maley et



Portions of this paper (including part of “Experimental Procedures,” part of “Results,” part of “Discussion,” additional Figs. 1-6, Tables 1-7, and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-3553, cite the authors, and include a check or money order for $6.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. Since the lysozymes which are the focus of this report are of the c type, the term “lysozyme” shall denote lysozyme c. The terms “cow” and “bovine” are used interchangeably to refer to domestic cattle of the species Bos taurus.

11617

11618

Stomach Lysozymes

al., 1979); the tryptic digestion was subsequently performed during 24 h a t 37 "C with an enzyme/substrate ratio of 1:50. Trypsin was pretreated for 16 h with 0.0625 M HCl a t 37 "C. Decitraconylation was carried out in 30% acetic acid for 2 h at 20 "C. The reduced and alkylated bovine lysozyme (15 mg) was also subjected to tryptic digestion (5 h, 37 "C) without citraconylation in 0.1 M ammonium bicarbonate with an enzyme/substrate ratio of 1:40. Digestion with S. aureus V8 protease of the reduced and alkylated lysozyme (15 mg) was carried out after solubilization of the latter in 0.1 M ammonium bicarbonate containing 1.5% sodium dodecyl sulfate (7 ml); after dialysis against 0.1 M ammonium bicarbonate containing 0.01% sodium dodecyl sulfate for 24 h at 4 "C, the protease (0.4 mg) was added and the digestion carried out for 24 h a t 37 "C; after a new addition of the protease (0.2 mg), the digestion was continued for 18 h at 37 "C. Chemical cleavage wascarried out by solubilizing reduced and alkylated bovine stomach lysozyme (15 mg) in 70% formic acid (5 ml), adding cyanogen bromide (140 mg), and allowing the reaction to proceed for 24 h at 20 'C; 30% acetic acid (20 ml) was then added and thesolution concentrated by lyophilization. Peptide Purification and Analysis-Filtrations on Sephadex G-10, G-25, G-50, and Bio-Gel P-60 (200-270 X 1.2-2-cm columns) were performed with 30% acetic acid as theeluant (12 ml/h). The peptides were detected a t 280 nm or by the fluorescamine procedure (Nakai et al., 1974) with a Jobin and Yvon JY 3D spectrofluorimeter. Preparative paper chromatography (Whatman No. 1) in n-butyl alcohol/ pyridine/acetic acid/water (15:103:12, v/v/v/v) or paper electrophoresis (Whatman No. 1; 50 V/cm; 45 min) at pH6.5 in pyridine/acetic acid/water (100:3.5:900, v/v/v) was the final purification step for several peptides. The amino acid composition of the peptides after total hydrolysis (5.6 M HCI containing 1:2000 2-mercaptoethanol; 18, 48, and 72 h; under vacuum) was determined with a Biotronik Autoanalyzer. Sequence Determination-Automated Edman degradation was carried out in a Beckman Sequencer 890 C for longer peptides by the 1 M Quadrol double-cleavage method (Edman and Begg, 1967) and for shorter peptides by the 0.2 M Quadrol single-cleavage method in the presence of Polybrene (Tarr et al., 1978). The phenylthiohydantoins were identified by thin-layer chromatography with chloroform/methanol (90:10, v/v) and neat chloroform as solvents and by high performance liquid chromatography with aWaters chromatograph (model ALC/GPC-204; pBondapak CIS column) employing the acetate system: 40 mM sodium acetate at pH 4.4 mixed with methanol in a ratio of 9 1 (v/v) for buffer A and in a 1:9 ratio for buffer B. The COOH-terminal amino acids were determined by digestion with carboxypeptidases A and B a t 37 "C for different time intervals presence of 2 mM in 0.1 M ammonium bicarbonate andinthe diisopropyl fluorophosphate; the digests were then analyzed on an amino acid autoanalyzer to determine released COOH-terminal amino acids.

bovine lysozyme for 24 h at 37 "C with carboxypeptidases A and B. Tryptic Peptides from Citraconylated Bovine Lysozyme and Their Alignment-As this lysozyme contains only 3 arginine residues per molecule, it was decided to submit the reduced and alkylated enzyme (20 mg) to trypticdigestion after citraconylation. The digest was decitraconylated and filtered on Sephadex G-25. Four peaks (CTl-CT4) were resolved and the four peptides contained thereinwere characterized. These four peptides add up to a total of 129 amino acids in the molecule and are in accordance with the arginine content. Peptides CT3, CT4, and CT2 were sequenced with an automated sequenator (Fig. 1):the last fragment corresponds to the COOH-terminal sequence of the bovine lysozyme, as it is devoid of a basic amino acid. Since CT3 and CT4 were contained in the NH2-terminal sequence of the bovine lysozyme, the alignment of the four tryptic peptides of the citraconylated enzyme is CT3 + CT4 -+CT1- CT2 (Fig. l). Cyanogen Bromide, Tryptic,and Protease Peptides: Completion of the Bovine Stomach Lysozyme Sequence-For peptide CT1 (Fig. 1) only a 17-amino acid NH2-terminal sequence was determined by automated analysis; an extensive study of diverse peptides encompassing the 75 COOH-terminal residues of CT1 was thus necessary in order to establish the complete sequence of cowstomach lysozyme. It was completed by examination of some of the peptides obtained from the reduced and alkylated protein by cyanogen bromide cleavage (CN peptides), by digestion with trypsin without citraconylation (T peptides), and by digestion with S. aureus V8 protease (SP peptides). Three peaks (CN1-CN3) were isolated by gel filtration of the digest obtained after cyanogen bromide treatment of bovine lysozyme, which contains only 1 methionine residue/ molecule (position 84,Fig. 1). Besides CN1 and CN2, the NH2- and COOH-terminal moieties of the protein, respectively, a third peak was obtained, CN3, containing two peptides which were formed by cleavage after tryptophan residues. Lability at tryptophan residues in general during CNBr treatment complicated use of this fragmentation method (see also Blumenthal et al., 1975; Braunitzer and Aschauer, 1975; Ozols and Gerard, 1977). Peptide CN3a permitted determination of a portion in the center of fragment CT1, while peptide CN3b encompassed the COOH-terminus of CT1 and all of CT2 (Fig. 1). RESULTS From the trypticdigest of bovine lysozyme, fivepeaks (TlCow Stomach Lysozymes Are of the c Type-The three, non- T5) were characterized after gel filtration. Five peptides sitallelic lysozymes that Dobson et al. (1984) purified from the uated in fragment CT1 were automatically sequenced. cow stomach mucosa were shown in the present study to be At this point, the entiresequence had been established (Fig. of the c type as regards molecular size, extinction coefficient, 1) but there was no overlap encompassing the junction of amino-terminal sequence, and reactivity with chitotetraose; peptides T l b and T5. We therefore characterized six fractions in addition, they areextremely closely related to one another (SP1-SP6) from the S. aureus V8 protease digest after gel in amino acid composition (see Miniprint andDobson, 1981). chromatography, with peptide SP2 being isolated from peak In these respects, they differ sharply from lysozymes of the g SP2. Its sequence (Fig. 1) allowed the alignment of tryptic The structure of fragment type, which appear to be present at very low levels in non- peptides Tlb, T5, and Tla. primary stomach tissues of the cow (Dobson et al., 1984). The most CT1 and therefore of bovine stomach lysozymewas thus abundant of the three lysozymes c, namely cow lysozyme 2, established (Fig. 1). Immunological Cross-reactions-Since polyclonal rabbit was subjected to detailed sequence analysis as follows. antisera are known to provide an approximate measure of NH2-terminal Sequence and COOH-terminal Amino AcidThe NH2-terminal sequence was established by subjecting 140 sequence relatedness among lysozymes c, when tested by the nmol of the reduced and alkylated protein to 46 cycles on a micro-complement fixation method (Benjamin et al., 19841, sequenator (Fig. 1). Lysine was the NH2-terminal aminoacid. we used this method to compare the stomach lysozymes of The different tryptic peptides contained in this 46-residue the cow and othermammals. Micro-complement fixation tests sequence were characterized again when the tryptic digests confirmed that the threelysozymes (1,2, and3) purified from cow stomach are very closely related to one another antigenwere studied. Leucine (0.2 residue) was the unique COOH-terminal ically. This is evident from tests made with antisera to COW amino acid obtained from digestion of reduced and alkylated lysozymes 1 and 2 (Table I). With both antisera, the immu-

Lysozymes

Stomach

Human Chicken cow

11619 Arg Ile Arg GlY Tyr

Met

Ar9 Arg His

Ala Ala Met

Asn

20 10 Lys-Val-Phe-Glu-Arg-Cys-Glu-Leu-Ala-Arg-Thr-Leu-Lys-Lys-Leu-Gly-Leu-Asp-Gly-Tyr-Lys-Gly-Val-Ser-Leu-Ala77777777777”7-77777777”777

CT 1

-%7557??F777’7777777777”7

Human Chicken cow

Met A1 a Val Ala Ala 30

A r9 Gln

Gly

Asn Phe

Phe

Ala Gly Asp Arg Thr 0 Asp Gly

Arg

40 50 Asn-Trp-Leu-Cys-Leu-Thr-Lys-Trp-Glu-Ser-Ser-Tyr-Asn-Thr-Lys-Ala-Thr-Asn-Tyr-Asn-Pro-Ser-Ser-Glu-Ser-Thr77777777777777777777

CT1

7

-

Tlb-

=

77777777777

Arg Tyr A rg

Gly

Human Chicken

Leu

Ser

Gly

Asn Ala Arg Asn Leu

At-9

Asn

60

cow

70 Asp-Tyr-Gly-Ile-Phe-Gln-Ile-Asn-Ser-Lys-Trp-Trp-Cys-Asn-Asp-Gly-Lys-Thr-Pro-Asn-Ala-Val-Asp-Gly-Cys-His-

+

CN3a

777777777777777

w

b ” T 5

777777-7777777777777777777

SP2 7777777777777777777777

Human Chicken cow

Arg Val ArgAsp Pro As P Thr Ala Ser Asn Asp Gly Asn 80 90 100 V a l - S e r - C y s - S e r - G l u - L e u - M e t - G l u - A s n - A s p - I l e - A l a - L y s - A l a - V a l - A l a - C y ~ - A l a - L y ~ - L y s - ~ ~ ~ - V ~0 ~ --~ ~~ ~ l- ~~~ ~- -

Leu

Ile Pro

Ala Ala

-

Leu Gln Asp Asn Leu SerSer

CN2

b4

7777777777777777777

7

“777-777777777 T1 a b

Human Chicken cow

Arg Asn Arg Arg Asn Arg

Arg Met Asn

4

7777”--77

Gln Asn Arg Lys Gly Thr

Arg Gln G1 n Gln Ala Trp Ile Arg

Gly Val Arg

110 120 Gly-Ile-Thr-Ala-Trp-Val-Ala-Trp-Lys-Ser-His-Cys-Arg-Asp-His-Asp-Val-Ser-Ser-Tyr-Val-Glu-Gly-Cys-Thr-Leu CT1

U

CT2

b

7777777777777

CN2

7777

“T2

h

-T3+

777777777-7777

t CN3b FIG. 1. Amino acid sequence of cow lysozyme 2. The first 46 residues were determined directly with a sequenator.The four tryptic peptides obtained after citraconylation of the onlysozyme the figure, are as indicated well as the various cyanogen bromide, non-citraconylated tryptic, V8 protease and peptides necessary for the -, residue determined by automated Edman degradation;0, deletion. The establishment of the sequence. sequences of chickenhuman and lysozymes are given at those positions where the To enzymes facilitate differ. discussion in relation to previously reported sequences, the residues are numbered according to the chicken sequence; due tomammalian the addition between positions 47 and 48 and the bovine deletion at 102, residue these numbers are out of register with those cow appliedand human to sequences. the actual 4

777777777777777777

Stomach Lysozymes

11620

TABLEI Immunological differences among ungulate lysozymes measured with antisera to cow lysozymes I and 2

cause deviations from its average rate of sequence change. Hemoglobin is the only other protein which has offered a similaropportunity. Wereferespecially to its loss of old functions (i.e. the bindingof organic phosphate, chloride, and Immunological carbamino CO,) and gain of a new function (Le. bicarbonate Lysozyme distance' binding) on the lineage leading from an ancestral terrestrial cow 2 cow 1 reptile to the aquaticcrocodilians. This functional shift repPure lysozymes resents anadaptively significant evolutionary response to the 0 cow 1 8 problem of blood acidity, which develops during prolonged cow 2 9 0 16 8 cow 3 periods under water, and canbe explained by five amino acid Stomach extracts' substitutions (Perutz etal., 1981). The question raised here is 7 2 cow' whether the lineage leading to crocodilian hemoglobins has 20 19 Goat experienced more sequencechange than havelineages retain24 16 Sheep ing the standard set of functional properties. The answer, 28 21 Bighorn sheep although apparently positive (Perutz et al., 1981), is ambigu39 29 Black-tailed deer 40 31 Axis deer ous because the time elapsed since the divergence of the 54 41 Roe deer crocodilian lineage from other hemoglobin lineages is great 67 56 Camel enough to allow not only multiple substitutions at many sites 75 70 Pig but also intergenic exchange events tooccur at thismultigene -70 -65 Horsed locus and thus obscure the record of point mutational diverThe immunological distances were determined with the quanti- gence (cf. Martin et al., 1983). In the absence of a clearcut tative micro-complement fixation method (Champion et al., 1974; cf. Miniprint). Immunodiffusion tests gave results consistent with the answer from this hemoglobin example, the case of ruminant lysozymes c is of particular interest. immunological distances. Extent of Evolutionary Change-As a firstapproach to For all stomach extracts the peak of the micro-complement fixation curve occurredat a dilution consistent with the levels of lysozyme finding out how divergent the cow lysozyme sequence is, we reported from activity measurements (see Dobson et al., 1984). calculated the number of amino acid differences between this 'The immunological distances for the cow stomach extract are lysozyme and others of known sequence (see Table 11);there commensurate with expectations, given the values above for the pure enzymes and the observation that cow 2 predominates in such ex- is a minimum of 38 amino acid substitutions and the one tracts, with lesser amounts of cow 1 and little cow 3 (Dobson et al., deletion (see below) from the other mammliansequences and at least 56 differences from the bird sequences. In terms of 1984). Values estimated from immunodiffusion reactions (cf. Prager and both this measure and the minimum number of base substiWilson, 1971; Prager et al., 1976) comparing the pig and horse with tutions needed to account for the aminoacid differences, the the axis deer. Technical problems prevented direct determination by cow lysozyme is not unusuallydivergent. Indeed, the cow micro-complement fixation. generally differs by a little more than primates do and a little does frombirds asregards minimal mutation nological distances among these three lysozymes are smaller less than the rat than those between cow lysozymes and those of sheep and distance. Phylogenetic analysis shows thatthe cow lysozyme is goats, which are the closest relativesof the cow tested. More distantlyrelated species of hoofed animals have stomach closely related to other mammalian lysozymes c (Fig. Z), in the same way that the species themselves are thought to be lysozymes showing progressivelygreater differences from cow related (Fitch and Langley, 1976); this implies that the selysozymes in their antigenic behavior. This is evident from comparing the immunological distances among lysozymes to quences have an orthologous relationship (Wilson et al., 1977). The phylogenetic tree in Fig. 2 suggests that the cow lineage the orderof branching of the lineages in theevolutionary tree has accumulated a modest number (30.8) of base changes for those species and to the times of divergence of those leading to amino acid changes, as compared to the rat(48.91, lineages from the cow lineage (see Miniprint;Fig. 6). Surprisingly, some non-ruminants (e.g. pig and horse) have stomach human (37), andbaboon (29.1) lineages. Thus, despitehaving lysozymes c that are aboutas closely related immunologically lost an old function and gained a new one, which affected its catalytic properties and possibly its resistance to pepsin (Dobto cow lysozyme c as is thecamel enzyme. Immunological distances between cow lysozymes c and those from primates and rodents aremore than twice as large as the maximum distances in Table I. An extremely weak reaction was seen with langur stomach and squirrelmonkey milk lysozymes andnoreactions were detectedwithpure human, baboon, and rat lysozymes in immunodiffusion tests with antisera to cow lysozymes. In a reciprocal comparison, an antiserum from one of four rabbits immunized with baboon lysozyme reacted very weakly with cow lysozyme. DISCUSSION

TABLE I1 Amino mid sequence differences and minim1 mutation distances among animal lysozymes The number of amino acid differences between any two lysozymes is given in the upper right-hand section of the matrix, while the minimal mutation distance appears in the lower left-hand section. Each addition and deletion has been counted as 1 amino acid difference and as one mutation in the computation of minimal mutation distances. ~

Lysozymes compared

~~~

Cow 2 Baboon Human Rat Chicken Duck Chachalaca

Adaptive Shifts and Sequence Euolution-It is important to 47 Baboon obtain a molecular understanding of one of the generalizations Human 48 emerging fromcomparisons of proteins, uiz that the rate of Rat 70 amino acid substitution in a given type of protein (e.g. lyso- 71Chicken70 76 75 zyme c ) is about the same along the various lineages leading Duck 1" to present day species of vertebrates (Wilsonet al., 1977). Our Chachalaca71 73 study of cow lysozyme may offer a n unusual opportunity to "From Kondo et al. sequenced. find out whether adaptively significant changes in this protein

39 33 14 45

41 14

68

69 76

55 37

51 76

83 77

58 50 52 58 26 30

57 49 52 55 21

56 50

55 57 27 29

35

(1982), one of five different duck lysozymes

,

rc Lysozymes

Stomach

11621

prolyl bond at positions 101-102. For the human, baboon, rat, and mouse, this is known from sequenceanalysis; for the horse (a close relative of the ruminants), this is evident from Boboon 144 end group studiesonthe two peptidesinto which horse Rot at 40 "C (Jauregui-Adell and lysozyme is cleavedbyacid Marti, 1975). From this, we infer that the ancestral mammacow lian lysozyme c probably had this acid-sensitive bond and FIG. 2. Treeshowing genealogical relationships among that itwas lost on the lineage leading to ruminants.A possible mammalian lysozymes of known amino acid sequence and the implication is that one or the other of these two mutations number of mutations needed to account for the divergence of these sequences from a common ancestor. This tree is based on may have beenfixed as the resultof selection pressure for the the parsimony principle, which means that it accounts for the se- ability of lysozyme to survive and function after exposure to quence diversity among the four mammalian lysozymes with fewer low pH (in the presence of substrates, fatty acids, pepsin, mutationsthan does any other branching order(cf. Ferris et al., 1981). surfactant mucins, andother chemicals) not only inthe A tree with the same branching orderas shown here and very similar stomach but, perhaps, in the duodenum aswell (see Dobson branch lengthsresulted when the minimalmutationdistances in Table I1 were subjected to phylogenetic analysis by the Farris (1972) et al., 1984). An additional feature of the cow lysozyme sequence that method. may be relevant to stability at low p H is its relatively low son et al., 1984) and stability in acid (see below), the cow content of aspartyl residues and amidegroups. The amide and lysozyme lineage appears not to have undergone accelerated aspartyl peptide bonds are acid-labile, although not to the evolution; cow lysozyme is not unusually far from the ances- extent of the aspartyl-prolylbond. In contrast to conventional lysozymes c, which have a n average of 23 residues (range, 20tral mammalian state in aminoacid sequence (Fig. 2). The immunological results (Table I)reinforce this view: the 28) of aspartate, asparagine, and glutamine, cow lysozyme 2 degree of antigenic difference between cow lysozyme and the has only 17 such residues. N e t Charge and Charge Distribution-On the basis of eleclysozymes of pig and horse are hardly greater than the distance from the camel to the cow. Yet, the camel is a ruminant trophoretic evidence, Dobson et al. (1984) suggested that the with a high stomach lysozyme level and a low pH optimum, ruminant lysozymes are relatively non-basic and that this while the pig and horse are non-ruminants with low levels of might be responsible for their low catalytic activity at neutral stomach lysozymes that do not functionoptimally a t low pH and high pH values under physiological salt conditions. In (Dobson et al., 1984). One possible implication of these find- agreement with this suggestion, from the sequence ofcow ings is that onlya small number of mutations was required to lysozyme 2 we calculatea net charge of zero at pH 7.65 produce a ruminant stomach lysozyme from a conventional (uncertainty, 7.40-7.83),3 which contrasts with thehigh poslysozyme itive charge borneby most lysozymes at this pH. The lysozyme. of known sequence that is most similar cow to 2 in net charge Location and Nature of Amino Acid Replacements-The positions at which the cow sequence has diverged are for the is thatof the baboon,which also hasa low pH optimum under most part unremarkable. There is an apparent amino acid physiological salt conditions(Dobson et al., 1984). The differsubstitution at only one of the 40 positions previously found ence in net charge between cow lysozyme and the convento be invariant in lysozymes c: aspartate for asparagine a t tional lysozymes c of rat, human, andbirds at pH8 is from 6 position 74. The residue at thisposition in chicken lysozyme to 10 units. Thisdifference is due more to an increase in the number of negative charges than to a decline in the number probably has no contact with the hexasaccharide substrate accordingtoSmith-Gill et al. (1984). Of the 25 positions of positive charges. A minimum of six mutations would thus where the cow enzyme has a unique amino acid or gap, 24 of be required to produce a lysozyme having a fully ruminantthem are likely to be either fully external or on the surface of like net charge from a conventional lysozyme. Assuming that cow lysozyme has the same general threethe molecule (Browne et al., 1969; Lee and Richards, 1971),if dimensional structure as that of chicken and humanlysozyme it is assumed that the three-dimensional structure is similar to thatof chicken and human lysozyme (Artymiuk and Blake, (Artymiuk and Blake,1981), we calculate that,at pH 8, every 1981). Of these, five positions (75,101,102,112, and 113) face4 of cow lysozyme bears more negative charges than does have been implicated among 30 contact residues. In contrast, the corresponding face in chicken or human lysozyme: averand -6.2, respectively. The number of two among ages of -8.7,-5.3, there are only12 positions (11external or surface, positive charges per face, in contrast, is less for the cow (9.5) contact residues)where eitherorbothprimate lysozymes than for the chicken or human (each11.9). From all aspects, (human and baboon) have an amino acid not seen in other vertebrate lysozymes; for the two rodent lysozymes (rat and then, cow lysozyme has less net positive charge and so will be mouse taken collectively) the corresponding value is 17 (15 less attracted than conventionallysozymes to the negatively external or surface, five among contact residues). This anal- charged bacterial substrate. This result fits with the explanation offered by Dobson et al. (1984) for the low catalytic ysissuggests that cow lysozyme mayhaveaslightlymore specializedoverallsequence to go withitspresumed new For this calculation we used the pK values of the chargeable function as a digestive enzyme. Possible Adaptive Significance of the Changes at Positions amino acid residues of chicken lysozyme given by Imoto et al. (1972). Our calculated PI value of 7.65 k 0.22 agrees with the value of 7.5 101 and 102-The only specific changes incow lysozyme that 0.1 observed by Pahud and Widmer (1982) for the major component we have succeeded in tentatively relating to its novel function (D) of calf stomach lysozyme by isoelectric focussing. are the deletion of proline a t position 102, along with the Six faces of the lysozyme molecule were considered front, back, adjacent substitution of glutamate for aspartate a t position left, right, top, and bottom, as defined by Smith-Gill et al. (1982). At 101. These two mutations eliminate the aspartyl-prolyl bond, pH 8, the side chain of glutamate 35 was assigned a charge of -0.95, which is more sensitive to acidhydrolysis at physiological histidyl residues +0.06, amino termini f0.33, all other carboxyl groups -1, and all lysyl and arginyl residues +1 (Imoto et al., 1972). The temperature than any other bondlysozyme in (Jauregui-Adell approximation was made that any charge-bearing amino acid residue and Marti, 1975; Landon, 1977; Inglis, 1983). The lysozymes contributed its charge to all faces on whose surface it appeared c of all five monogastric mammals tested have an aspartyl- according to Smith-Gill et al. (1982). Humon

11622

Lysozymes

Stomach

activity of cow lysozyme c at high pH. Acknowledgments-The excellent technical assistance of Ly @ a n Le is gratefully acknowledged. We thank S. J. Smith-Gill, C.R. Mainhart, and R. J. Feldmann for providing crystallographic outlines of chicken lysozyme and S. M. Beverley, R. D. Cole, W. S. Davidson, H. Fujio, M. F. Hammer, P. V. Hornbeck, J. Jauregui-Adell, M. McClelland, J. A. Rupley, V. M. Sarich, E. L. Smith, S. J. SmithGill, C.-B. Stewart, and P. Van Soest for helpful discussion. REFERENCES Artymiuk, P. J., and Blake, C. C. F. (1981) J. Mol. Biol. 152, 737762

Benjamin, D. C., Berzofsky, J. A., East, I. A., Gurd, F. R. N., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, A., Prager, E. M., Reichlin, M., Sercarz, E. E., Smith-Gill, S. J., Todd, P. E., and Wilson, A. C. (1984) Annu. Rev. Zmmunol. 2,67-101 Berthou, J., Lifchitz, A., Artymiuk, P., and Jolles, P. (1983) Proc. R. SOC.hnd.B Biol. Sei. 217,471-489 Blumenthal, K. M., Moon, K., and Smith,E. L. (1975) J. Biol. Chem. 260.3644-3654

Braunitzer, G., and Aschauer, H. J. (1975) Z. Phys. Chem. 356,473474

Browne, W. J., North, A. C. T., Phillips, D. C., Brew, K., Vanaman, T. C., and Hill, R. L. (1969) J.Mol. Biol. 42,65-86. Champion, A. B., Prager, E. M., Wachter, D., and Wilson, A. C. (1974) in Biochemical and ImmunologicalTaxonomy of Animals (Wright, C. A., ed) pp. 397-416, Academic Press Inc. Ltd., London Dobson, D. E. (1981) Ph.D. dissertation, University of California, Berkeley Dobson, D. E., Dayan, E., and Wilson, A. C. (1979) Fed. Proc. 3 8 , 674

Dobson, D.E., Prager, E. M., and Wilson, A. C. (1984) J.Biol. Chem. 259,11607-11616

Edman, P., and Begg, G. (1967) Eur. J. Bwchem. 1,80-91 Farris, J. S. (1972) Am. Nat. 106,645-668 Feeney, R. E., and Allison, R. G. (1969) Evolutionary Biochemistry of Proteins, John Wiley and Sons, New York Ferris, S. D., Wilson, A.C., and Brown, W. M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,2432-2436 Fitch, W. M., and Langley, C. H. (1976) Fed. Proc. 35,2092-2097 Gavilanes, J. G., Gonztilez de Buitrago, G., Martinez del Pozo, A., PBrez-Castells, R., and Rodriguez, R. (1982) Znt. J. Pept. Protein Res. 20,238-245

Imoto, T., Johnson, L. N., North, A. C. T., Phillips,D. C., and Rupley, J. A. (1972) Enzymes, 3rd Ed., (Boyer, P. D., ed) Vol 7, pp. 665568, Academic Press, New York Inglis, A. S. (1983) Methods Enzymol. 91,324-332 Jauregui-Adell, J., and Marti, J. (1975) Anal. Biochem. 6 9 , 46&473 JollGs, J., van Leemputten, E., Mouton, A., and Jollcs, P. (1972) Biochim. Biophys. Acta 257,497-510 Jolks, J., Ibrahimi, I. M., Prager, E. M., Schoentgen, F., Jolles, P., and Wilson, A. C. (1979a) Biochemistry 18,2744-2752 Jollb, J., Schoentgen, F., Crozier, G., Crozier, L., and Jolles, P. (1979b) J. Mol. EVOL 1 4 , 267-271 Jung, A., Sippel, A. E., Grez, M., and Schiitz, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,5759-5763 Kondo, K., Fujio, H., and Amano, T. (1982) J. Biochem. (Tokyo) 9 1 , 571-587

Landon, M. (1977) Methods Enzymol. 4 7 , 145-149 Lee, B., and Richards, F. M. (1971) J . Mol. Biol. 55,379-400 Maley, G. F., Bellisario, R. L., Guarino, D. U., and Maley, F. (1979) J. Biol. Chem. 2 6 4 , 1288-1295 Martin, S. L., Vincent, K. A., and Wilson, A. C. (1983) J . Mol. Biol. 164,513-528

Nakai, N., Lai, C. Y., and Horecker, B. L. (1974) A d . Biochem. 5 8 , 563-570

Osserman, E.F., Canfield, R. E., and Beychok, S. (eds) (1974) Lysozyme, Academic Press, New York Ozols, J., and Gerard, C. (1977) Proc. NatL Acad. Sci. U. S. A. 7 4 , 3725-3729

Pahud, J.-J., and Widmer, F. (1982) Biochem. J. 201,661-664 Perutz, M.F., Bauer, C., Gros, G., Leclercq, F., Vandecasserie, C., Schnek, A.G., Braunitzer, G., Friday, A. E., and Joysey, K. A. (1981) Nature (Lond.) 291,682-684 Prager, E. M., and Wilson, A. C. (1971) J. Biol. Chem. 246, 70107017

Prager, E. M., Fowler, D. P., and Wilson, A. C. (1976) Evolution 3 0 , 637-649

Smith-Gill, S. J., Rupley, J. A., Pincus, M.R., Carty, R.P., and Scheraga, H. A. (1984) Biochemistry 23,993-997 Smith-Gill, S. J., Wilson, A. C., Potter, M., Prager, E. M., Feldmann, R. J., and Mainhart, C. R. (1982) J. Zmmunol. 128,314-322 Tarr, G. E., Beecher, J. F., Bell, M., and McKean, D. J. (1978) Anal. Biochem. 84,622-627 White, T. J. (1976) Ph.D. dissertation, University of California, Berkeley Wilson, A. C., Carlson, S. S., and White, T. J. (1977) Annu. Rev. Biochem. 46.573-639 Additional references are found on p. 11625.

Stomach Lysozymes SupplementaryMaterialto

11623

"StOmCh Lyyrozyner o f Rumnantr 11. m i n a A c i d Sequence o f C o r Ly101yne 2 and l m n o l o g i c a l COmparisOns withOther Lyrozyner"

P l e r r e J o l l l s . Franc.oire Schoentgcn, Jacqueline J o l l h , Deborah E. Dobson. E l l e n M. Pragcr. and Allan C. Y i l l o n EXPERIWNTALPROCEDURES

9 0.6

Al e r p r i m n t a l pmceduresdescribed i n t h e S u p p l m n t a r y M a t e r i a l a r e those employed by theBerkeleylaboratory.Fordetermination Of amino acidConpositionl,amino-teminal requencing, and g e l f i l t r a t i o n , p m c e d u r e s which were Conducted i n Paris as w e l l d l i n Berkeley, details of the methods used bythe J o l l l l l a b o r a t o r y appear i n the main t e x t . Electmphorerlr

cc

Denaturinggelelectmphorerirthrough151polyacrylamidewith sodium dodecyl s u l f a t e as thedenatulant was c a r r i e d Out as describedby ODblen e t a l . (1984). F a l l a i n g t h e pmcedure Of L d e m l i (1970). a standard curve r e l a t i n g s i r e t o e l e c t m p h o r e t i c m b i l i t y f o p p m teins of knan mlecular weight was constructed and used t o e r t i m t e t h e m l e c u l a r w e i g h t Of c a stomach lyrezyner.

0.4

0.2

Gel F i l t r a t i o n A Sephadex 6 7 5 c o l u m (57 x 2 m , 180 n l ) e q u i l i b r a t e d i n 0 . 2 1 a c e t i c a c i d was C a l i brated with pmteins O f knan mlccular eight. Sanpler o f p u r i f i e d c a l y s o z m 2 were and m l e c u l a r we1 h t was calculated fmm the chmmtagraphed to detennlnc elution position. The data were analyzed as describedbyFischer p19691: f o r each p m t e i n . standard CUIYC. t h e r e t a r d a t i o n c o e f f i c i e n t K was c a l c u l a t e d as K,, = (V - W l/(Vt-V,,l, where We = malured e l u t i o n valune, Vo = v o i d uolune. and Vt = t o t a l m l u m $ e l 8 m .

I 20

10

I 40

,

1

I , I 80 100

I

60

Molecular Weight

Fig. 1. Molecularweightdetermination o f coy Stomach l y l o z y m l baled on denatunng gel electrophoresis. A standard CUPW was c o n s t w c t e d i n Mhlchthe m b i l i t y o f each p m t e i n i s p l o t t e d againstthe m l e ( r e l a t i v e t o c y t o c h m n r c i n 151denaturingpolyacrylamidegels) cularweight. The r e l a t i v e m b i l i t i e s o f c a stomach lysozynes a r e S h a n a s open c i r c l e s . P m t e i n s t a n d a r d s i n Order o f increasing size yere c y t o c h r n r C . h m n lyrozyne C . chicken g a m globulin,glyceraldehyde-3l y s o z m C . ribonuclease,myoglobin,chymotrypsinogen, phosphate dehydmgenase, alcoholdchydmgenare,glutamic dehydrogenase, catalase,bovine s e w albumin, and phosphorylase a .

Extinction Coeffldent Pumfied, lyophilized lysozymes were weighed, dissolved i n 0.05 H radium phosphate. pH 7, and the absorbance a t 280 nm rrcllured w i t h a Cary r p e c t r o p h o t o n e ~ y . Chicken l y s o 2 y m E served as a c o n t m l . and a l l masured valuer were n o m l i z e d to an E Z w n m o f 26.7 f o r chickenlylozyme c (Amheim e t al., 1969, and referencestherein). Amino Acid h a l y s i s Lyso2ynees were subjected to h o successive r m n & Of r e d u c t i m and c a r b o w t h y l a t i o n based on t h e pmcedure described by Amheim e t a l . (1969). The denaturant was 5 M guanidine nll a n o n i v n h y d m r i d e r a t h e r t h a n hydmchloride and the solutions were dialyzed against 1 againstwater and thenlyophilized. The p m t e i n s were hydrolyzed under vacum i n conrtant8, or 72 h. and t h e wiM a c i d c o w s i t i o n s were bte? b a i l i n g HCl a t 105-110° C f a r 24. 4 mnedwith a Beckran 121 aUt(YMtiC amino acidanalyzer.Serine and threonine were d e t e n i n e d i n t of hydmlyrir. the valine and i s o l e u c i n e v a l u c ~ were taken f m by e x t r a p o l a t i o n t o zero t the 72-h h y d m l y r a t e r . and l / Z - c y r t i n 11%determined as S - c a r b o x y r r t h y l c y $ t e i w . Tryptophan was measured by the pmcedure of Edelhoch (19671 w i t h 5 II guanidine hydmchloride IS the denaturant. Chicken lysolyne served a s a c o n t m l .

0.6

I

I

I 20

40

I

1

0

h i n o - T c m i n a l Sequence h a l y r i r

The method o f Edlan (19701 as describedbylbrahimi (19773 was used. Degradations were The phcnylthiohydantoinderivatives here hydmc a r r i e d o u t on 100-200 n a n m l e r o f p m t e i n . lyzedtothefree amino acldr in constant-boiling HCl a t 140° C f o r 15 h under Y I C Y Y ~ . The amino acids were i d m t i f i e d w i t h a Beckman automatic amino acid analyzer as described above. Chicken l y r o z p was i K l u d e d as a c o n t m l . Carbohydrate h d l y s i s S u f f i c i e n t m t e r i a l was used t o d e t e c t me =si& OP less Of sugar p e r m l e c v l e O f p r o t e i n . The pmcedures outlined byAmheim e t a l . (19691 yere f o l l a r e d t o d e t e c t n e u t r a l sugars and s i a l i c acid; amino sugals were detected i n acid-hydrolyzed r w l e s m i n g a Beckman amino acid analyzer. with glucosamine a s the standard. htisera,htigenr,

t

and I m n o l o g i c a l Wetho&

The r a b b i t a n t i s e r a t o b a b m and h m lysozymes (Hanke e t a l . . 1973) and r a t l y s o z m (Prager e t 11.. 19781 have been describedbefore. h t i s e r d t o cmr s t o m c h lyro-r were Prager and Y i l l o n (1971). Two g m p s O f f o u r h t c h pmduced e l l e n t i a l l y by the mthed of B e l t e d v a t b i t s were i m n i z e d . one w i t h p u m c a lysozyme 1 and the Other with PUR c a l y s o z p 2 : on day 1. each r a b b i tr e c e i v e d 0.2 "J O f lysozyme inwpplerrntedFreund'scanplete were begun on days 47.131,192. and 235; adjuvant. Serles o f intravenousboosterinjections each r a b b i t r e c e i v e d 0.2 "J O f lysozyneperinjection. The a n t i s e r a used i n t h i s work were fm post-b0olt bleeding$ t a k e n a f t e r 7 m t h r Of i m n i z a t i o n . The f o u r i n d i v i d u a l a n t i s e r a were pooled a s described by C h w i o n e t a l . (19741. Far use as antigens i n b o t h a i c m - c m Plenent fixation and i m n o d i f f u s i o n t e s t s . e x t r a c t s o f r t a n a c h and othw tissues containing la concentrations of lyrozyne were rmtimr l y o p h i l i z e d and redissolved i n i s o t r i s buffer (Champion e t d l . , 1974; Hornbeck and Y i l r m , 1984). Imunadiffus10n was done as described by Prageer e t a l . (1976). To needsure antigen C M centrations. wells ( 2 m i n dianeter),placed 2 m apart. m c e i w d u n d i l u t e d a n t i r e r v m 07 various dilutions O f a n t i n To detect weak reactions between antigen and antilerum.larger wells (5.5 m l n dianetery*;re s m t i m s used. Quantitative micre-conplemnt fixation (nC'F1 was done I S described by manpion e t a l . MC'F assay i s given i n t e r n o f innuno(1974). The degree Of a n t i g c n i cd i f f e r e n c ei nt h e lagicaldistance.which i s equal t o 100 t i m s t h e l o g O f t h e f a c t o r by whichtheantisew. a conplant fixation concentration must be r a i s e d f o r a heternlogousantigentoproduce curve *hare peak height i s equal to that pmducedby t h e h m l o g c m a n t i g e n ( t h e innunogen]. lysozyneribanucleare.azurin,qvoglobin.and serum Forseries Of theglobularpmteins albumin(0enjamin e t al., 19841, a l i n e a r r e i a t i o n s h i p has been observedbemeenpercent amno ac7d sequence difference and i m n o l o g i c a l d i s t a n c e , w i t h a correlation degree of a n t i g e n i c Coefficient Of about0.9 betwenthere parameterr.Besidesestimating difference by comparing peak heights, we estimatedantigenconcentrations by c n p a r i n g t h e antigendilutionsrequiredtoget peak f i x a t i o n .

01

I

I

I

60

Molecular Welght

The I n d i Fig. 2 . Ilolecularweight of c o 1 stmuch l y l a l y m 2 baled on gelChrmtogrdphy. cated l i n e was f i t t e d by eye t o t h e s o l i d c i r c l e r . which represent the valws observed f o r P m t e i n s Of k n m mlecula).weight: c y t o c h m c,chickenlysozyne C . ribonuclease,chymand hman 5ery.l albumin. The value Of theretardationCcefflcient trypsinogen.ovalbumin, f o r c a l y m z y m 2 i s s h a n as an open c i r c l e .

acid

Cow stomach

hino

RESUTS Physical and Chemical Characterization O f Ca S t m c h L y s o z y m l Molecular Yeight

Total

1

2

7.8 ( 8 ) 16.5 (15) 7.9 ( 8 ) 11.4(111 11.2 (11) 2.3 ( 2 ) 9.1 (9) 10.3 (10) 9.8 ( 9 1 (11 1.1 4.8 (51 9.1 ( 9 ) 4.6 (5) 2.1 121 2.3 ( 3 ) 9 . 8 (12) 3.2 ( 3 ) 6.3 ( 6 )

7.8 (8) 16.8 (15) 8.5 (81 12.9 (131 10.3 (10) 2.2 ( 2 ) 8.0 ( 8 ) 10.1 (10) 10.0 ( 9 ) 1.0 (11 4.7 ( 5 ) 9 . 1 (9) 4.5 (51 2.1 ( 2 )

129

Deternlnation

Of

2.3

Cow m l k b

ChlckenC

3 6

22-23 8

8 14 9 11 5 6 2 10 5-6

7 7

(3)

9 . 8 (12) 3.2 ( 3 ) 6.1 ( 6 ) 129 129

7

131d

(81 (21) (7) (la)

(5) (2) (12) (12) (6) (2) (61

la!

(3) (3)

(1) 5.0 ( 6 ) 10.2 (11) 6.0 ( 6 )

10 15 1 154

the Sequence of Cow StomachLysozyme

7.8 20.6 6.6 9.1 5.4 2.0 10.8 11.6 6.2 1.9 5 6 7 7 2.7 2.8 0.7

2

Stomach Lysozymes

11624

TPgLE 2

Pmpertler O f the tryptic peptlder O f reduced. a l k y l a t e d . and C i t r d a n y l d t e d b o v i n eI t m u c hl y l o z y n c :m i n oa c i dc o r p s i t i o n . R , c a t pH 6.5. and y i e l d Valyes are baled on h y d m l y r i r f o r 1 8 h . The nuoher o f ms!dueS perpeptideis given ? n parentheses t o thenearest Integer. Values & t a m e d by l i n e a r e x t r a p o l a t l o n t o z c m time ISer and T h r l o r a f t e r 48 and72 h O f h y d m l y r i r were taken i n t o Conslde r a t i a ni na r r i v i n ga tt h ei n t e g r a lv a l u e s . 112-Cystine was determined a s S-carboxyw t h y l c y r t e i n e . Tryptophan was i d e n t i f i e d by t h et h r l i c hr e a c t i o n and duringthe m = 0 f o r Gly. + I f o r Arq, and -1 f o r CvsteiCacid. a u t w t e d degradation. NO, n o t determined.

W

V Z W

V

In W

Peptide

(r

Total

0

CT2

3 LL

100

200

FRACTION NUMBER Fig. 3. F l l t r a t i o n on Sephadex 6-25 ( 2 M x 2 cm) O f t h e t r y p t i c d i g e s t o f 20 mg of reduced. alkylated. and citraconylatedbovineItaMCh l y r o z p . The e l u e n t was 302 acetic a c i d and t h e f r a c t i o n s i z e 2 m l .F l u o r e r c e n c e( a r b i t r a r yu n i t r l *ds measured by the emission at 480 m f o l l o l i n g e x c i t a t i o n a t 390 m.

Aspartic acid Threonine serine Glutamic a c i d Pmline Glycine Alanine Valine 112-Cystine Methionine Iroleucinc Levcine Tyrosine Phenylalanine Lyr ine Histidine Alqininc WpWhan Total Rf tbb?lity, m

Yield (I) LOCaliZatim. relid"= .y*crr

CT1

CT3

11.5 (13) 6.2 ( 7 ) 9.0 (11) 6.2 (7) 2.0 ( 2 ) 6.0 (71 7.5 (9) 5 . 0 I61 4.8 (6) 0.8 (1) 3.8 ( 5 ) 7.0 (7) 4.0 I41 0.7 (11 10.1 (11) 2.0 (2) 1.0 ( I ) + (61 106

CT4

2.0 (2)

15 8 13 10 2

1.0(1)

2.0 (21 0.9 (1)

1.0 (1)

1.0I11

1 . 0 (11

8

0.9 (11 1.6 (21 0.9 (11

10 9 8 1 5 9 5 2 12 3

0.9 I 1 1 1.0 I l l

1.0 (1) 0.8 ( 1 1

1.0 ( 1 1 0.6 I 1 0.8 (11

1.0 (1) 0.9 ( 1 )

3

1.0(1)

6 13

NO

5

0.35

5

0.36

129

0.30

ND

-0.58

30

42

61

50

11-116

117-129

1-5

6-10

4.40

0

TABLE 3

R and valuer. and y i e l d so ft h e cyanogen b m i d e h i n oa c i dC m w s i t i m l , Of RdlCed and alkyl&bovine s t m c h l y ~ o z Values ~ . were m a w r e d a$ described i n Table 2. k t h i m i n e lds i d e n t i f i e d as h m r c r i n e . NO. notdetemincd.

fra-ntl

peptide

Total

Peptide

hino acid

0

LL

n

0

012

Total

85

14

Rf mbility. m Yield (X) Localizatim. rrlidue nu-rr

NO NO

011 +

012

M3a

CN3b

22

18

50 100 FRACTION NUMBER

The t r y p t i c d i g e s t O f noncitraconylated. reduced, md a l k y l a t e d b v i n e l y r o z y r r was C h m t o g l a p h e d on Sephsdex 6-25 (Fig. 5 ) . F i v e peaks IT1 t o T51 e r e studiedandfive ND and 6. k l w ) . T h e e Of these peptidesderived frm w i t h i n CTl sequenced (cf.Tables4 peptides r e first p u r i f i e d by p r e p a m t i e p a p r chmnutography ITla and T l b l or e l e c t m lm because two Of i t s p h o r e l i l ( T Z ) . Peptide 15 was g r e a t l y retarded 011 t h e Sephahx u seven resi&s a r e t y p t o p h m r . The S t q A ~ l a o r r w r-w V8 protease digest of the reduced and a l k y l a t e d l y s o z y r was likewise ch-tographed M a 270 x 1.4 M c01.n Of Sephadex 6-25; peptide SP2 was isolated, characterized. and sequenced as m w r t e d i n t h e main t e x t and I " Tables 1 and 6 k l w

"

011

50

1 0 0

FRACTION NUMBER Flg. 5 . Filtration On SephadexG-25 (270 X 1.4 an) o f t h e t r y p t i c d l g e r t Of 15 mg O f reduced and alkylatedbovine r t m c h l y s o ~ The ~ . eluent was 301 a c e t i ca c i d and t h e f r a c t l o n 11ze 2 m l . F l u o m r u n c e ( a r b i t r a w u n i t r l was nearumd bytheemillion a t 469 nm f a l l a , n g e x c i t a t i o n a t 390 nn.

Aspa-ic acid Threonine Serine Glutamic a c i d Pmline Glycine Alanine Valine 112-Cystine kthionine Isoleucine LCUC7"e Tymrine Phenylalanine Lyllne Histidine Ayinine

0.7I11 1.9 (21 2.0 (2) 0.8 ( 1 ) 1.1(11 1.7 121 1.6 (2) 1.8 ( 2 ) 0.9 (11 0.7 (11 1.0 ( 1 1 1.0 (11 1.0 (11

4.1 1 4 ) 1.3 (2) 3.2 (41 1.8 I 2 1 0.8 (1) 1.0 (11 1.0 I 1 1

Vield

(:I

0.20 -0.38 30 69-90

1.0(1) 1.2(11

0.9 (11 1.8 (21 1.8 121 0.7(11

1.6 ( 2 1 0.6 (11 1.0 (11

16

21 0.25 -0.27

1.0(11

6.0 ( 6 1 1.8 I 2 1 3.5 I41 1.9 I21 1.0(11 2.7 ( 3 ) 0.8 (11 1.6 (21 2.6 (31 1.1(21

0.8 (11 I21

+ (21 22

Localization. reridue nynbe~

2.1 I 2 1 1.0(11 0.8 (11 2 . 0 (2)

1.6 (2)

.. .

Rf Poblllty. m

an e w l a n a t i m of why the t o t a l n h e r

1.5 (2)

T7YDtOl)hm

Total

20 112-129

63-84

the 1 0 C a l i l a t i o n .

fm+ka+m

4.2 (41

0.15

0

20

62 85.129'

ee the t e x t wrtim o f Pe ti&Pro m i e r f o r If r e s i d u e s d i f f e r r

0.10

-0.04

56 1-84'

129

NO

15

0.25 0

1.0 (11 1.0 (1) 1.0 (1) 4 0.12

+0.68

0 . 6 (11 1.6 (21 1.0 ( 1 )

+ 121 7 0.14

33 NO

0

33

16

22

43

30

42-61'

97.112'

113-116

62-68

50-82

Stomach Lysozymes

11625

Automated E d n a " Oegradation Table 5 d e t a i l s the C O Y ~ Saf ~ det cm i nat i on by autorated reqwntial degradation.

Of the flrrt 46 residues

Of

c w lyso2ym Fig. 6. Evolutionary relatlonshlps of rumlnantr and related ungulates. Divergencetimes a* bared on organlsnal (Romr, 1966; NOvaCek. 1982; Savage and Russell, 1983) and blochemica l( C a r lr o ne td l. . 1978; V. H. Sarlch and A. Bennett personal c o m n i c a t i o n ) evidenre

TABLE 5

redlced and alkylatedbovinertomchlysozym

Automated w q u e n t i a l degradationof

1 2 3 4

Lys VI1 Phe Gl"

7 8 9 10 I1 12 13 14 15 16 17 18 19

Gl" Le" Ala A q Thr

94 98 87 62 18 NCd 48 77

: 3.

Le"

20 21 22 23

31

50 10

32 33

54

35

34

22 27 21 14 19 15 12 14 12 12 16

NC 13 15 4

Ala Arn Tlp Le" CYS Leu Thr LYS Trp Gl" Scr

30

ut

Lyr Lys Leu Gly Leu Asp GlY Tyr LYl Gly Val

ser Leu

24 25 26 27 28 29

36 37

scr

18

TY? Arn Thr LYS Ala Thr Arn Tyr Am

39 40

si

42 43 44 45 46

3 16 NC 18 NC

3 2 6 NC NC 1 1 IC 1

3 WC 0.5 1

0.3

'The phenylthiohydantoin amino acids e m i d e n t i f i e d by thin-layerch-tegnphy high perfoomncc l i q u i d chmmtography ( H ) . bRepetitive yield.

(1) and by

DlSCUSSlOW Phylogenetic Tree for Manmalian L y m ~ y m s

To b u i l d t h e t r e e r h a n i n t h e m i n t e x t , r e l a t i n g t h e f o u r mmlim l y s o z y m ~Of k n a n sequence (Fig. 2 ) . we used t h r e e h ig h ly d ive r p e n t b ir d lys0 2 m s c I S an outgmup. Although 79 mim acidpositions a r e variable a n n 9 these seven lyso2ymsonlythe 15 posit i o n s l i s t e d i n Table7 pmved to be relevant as regards d e t cm in in g t h e &de7 o f branching ofthe m m l i a n lineages by the parsimony nethod. I n each of these 15 cases the n-r Of w t r t i m s r e q u i r e d t o account for the amino acid differences dcpcnded an the order O f branching i n the tm (Table7). The mrt P a r s i m n i w s tree (A i n Table 7) accounted forthe amino aciddiffemncesatthese 15 positionswith 48 nutations a t theMAlevel. The a l t e r n a t i v e trees (8, C . and 0) d i f f e r i n branching order and required f m 49 t o 56 w t a t i m s (Table7). A t each Of the m i n i n g 64 O f the 79 variablepositionsthe nu*r O f Ilytdtions required to accwnt for the amino aciddifferences was i n d e p e n h t o i t h e t r e e topolapy (A, B . C, o r 0).

94%.

'Cyrtelne was recovered as S - C . r b O X ~ i ~ t h y l C y ~ t e i n L . %C.

notcalculated.

TMLE 7 PhylogmetlCally infomtive

Thirteen peptides w m sequenced i n the course o f determination Of t h e i d e n t i t y and order o f a l l 129 amino acidsof co* l y r o v c 2. Table 6 . i n a f ashi m anal ogou~ t o Table 5. The r e p e t l t i v . y i e l d s Obtained details the COWSL of E b v n h g r a d a t l m ofthesepeptides. Of the m i n t e x t and Tables during aUtWbatlC Iequcncin9 Of the lmwr peptidCS (Cf.Fig.1 2-4 here) were as f o l l m : 0 1 . 76%; M 2 , 8 8 I : Tla. 89%; Tlb. 751.

3

Step

Yield

5

Leu

6 7 8

Gly Le" Asp Gly

9 Tyr 10 11 12 13 14 Leu 15 16 I7

CTZ--0 1 His 2

3

Tyr

Thr Leu L n LYS

LYI Gly Val

Ser Ala

h n

Asp YII scr

6

Ser

40 45 50 35 40

3

Asp Ile Ala

30 12 21 7 5

6 1 4 3 0.5

52 47 45 9 5

GI" Gly Cy$

Thr Leu

32 21 14 10

2 4

03-50 nml LYI

1 2

3 4 5

VI1 P C Glu Airg

28 35 20 16 8

04-40 nml 1 2

3 4 5

Cyr Gl" Leu Ala Arg

14 28 25 20 6

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 20 21

22

Lyr

Ala Val Ala cyr Ala

Lyr Lyr

Ilc Val

Ser Glu 61" Gly Ile Thr Ala

M31-30 mml 1 Trp 2 Cyr 3 Am 4 Asp 5 GlY 6 Lys Thr 7 8 Pm 9 As" 10 Ala 11 Val 12 Asp 13 Gly 14 Cys 15 H1I 16 Val M3b--90 n m l 1 Lys ser 2 3 His 4 Cyr 5 A q 6 Asp 7 His 8 Asp 9 VI1 10 scr 11 ser 12 Tyr

36

3 2

13

2 1

2 12 12 10 15 12 12 5 7 7 12 8 4

2 1 1 1

60

22 45

30 25

36 30 37

35 4 2 30

Tla-" 1

2 3 4 5 6 7 8 9 Val 10 11 12 13 14 15 16 17 18 19 20 A11 21

Vm Am A11 Val Asp Gly cyr His

2

3

9

Cy* Ser

4 4

Glu Leu Net Glu

11 3 4

As"

2

Alp Ile

4

Lys

0.5

3

As"

4 5 6 7

Tyr Am

PIO ser scr Glu ser Thr Asp Tyr Gly Ile Phe Gln

Ile ser LYI

scr

5 6

Glu Gln GlY r1e Thr Ala Trp VI1 Ala Trp Lys

13 14 15

73-40 1

rnl Ser

15-70

m o l

Yield

1

2 3 4

2 3

5 6 7 SPZ-70

48 13 28

35 25 15 4 2 14 2 9 8 4 4 4

3 2 1 0.2 2

Thr

TQ Tlp cyr Asn Asp Gly Lys

25 4

6 5 2 2 0.3

3 1 2 2 0.5

0.2

12

Ser

3

Asp Tyr GlY Ilc Phe

4 5

6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

20 15 20 17 16 5 2

nml

1 2

G " l Ile A m Ser Lys Trp Tro

Cy; Am Asp GlY

Lyr

Thr Pm Am Ala VI1

49

62 73 85

98 112 113 117 124 128 129 Total

20 25

I2

-1

Thr

4 7 8 9 10 11 12

16 13

4

1 2

10 11 1 12 13 14 15 16 17 18 Am 19 20 21

LYS 11e Val

23

scr

Tlb--103

8 9

mol

1

5 14 15 24

It

#A

12-40

" r n l Thr

22

Ala

Step

Yield

M3b (continued) 13 23 Val 14 15 61" 15 9 61y 16 6 Cyr 17 Thr 1 3 18 Le"

24 38 35 28 17 16 16 5 10 7 7 6 5 1

M

Step

49

1

As"

36 Val

Gl"

2

24

4 5 7 8 9 10 11 12 13

72

89

" n v l Asp

Yield

M2--90 r n l

01-110 nm1 1 2 3 4

IL

3 5 4 6 3 2 5 4 1 3 4

34

A u t w t e d Sequentialdegradatim o f p c p t i h S o f b i n e I t W C h lpazyne. The peptides arc i d e n t i f i e d I S t o l o c a t l o n i n themolecule i n Fig.1 O f the mi" t e x t and i n Tables 2-4 here. The n&r of nanmler rajected to degradation i s i n d i c a t e d n e . The phenylthiohydantoin u i m acids were Identii w d i a t e l y d f t w thepeptide f i e d and quantitated by high p e r f o m n a l i qui d chm ut ogr aphy. Y i el ds a r e given i n iA ~,, amino acid. n a n a o l e r . Cysteine was recovered as S - C ~ r b O O I . n i ~ t h y l C y ~ ~ A

M

awn9 mmm1 and b i r d l y s m y m s

15 21

TlBLE 6

Step

amino acid differences

10 11 42 40

M

36 34 27 24 19 4 13 8 7 7 5 6

3 4 0.8 1

0.5 1 1

2 4 4 4 4 4 3 5 2 3 4

3 5 3 6 4 4 5 5 2 2 3

3 5 4 6 4 4 5 5 2 3 4

2

3

3

3

2 3 1

1 4 2

2 4 2

2 4 2

48

49

53

M

'Tree A i s r h m i n Fig. 2 O f the .ai"t e x t . Tree 0 d i f f e r s i n branching order by allyingtheprimate and c a lineages n r t closely. Tme C associatesthe r a t and COW l i m a g e r mort ClOSelY. T m 0 a l l u ~ sa three-way s p l i t among t h ep v i m t e rat and CDI l i n a g e s . A11 f W r treespreserve thebranching order r h a by J o l l h ' e t a i . (1976. 197%) f o r t h e b i r d l y s o z y ~ r ~ . i n d i c h thehrck and chicken l i ~ a g eare ~ mre c l o s e l y r e l a t e d than e i t h e r i s t o the chachalaca lineage.

'ma.

ChaChalaCa.

The n e x t step u s t o assign the w t a t i m l a t each o f the 79 positions t o p a r t i c u l a r examlineages m t r e e A. At scam p o sit io n s,t h is was a s i w l c task, a s pointedoutfop ple. i n the r a i n t e x t f o r the deletion Of p m l i n e a t p o s i t i o n 102 On the CMI 1;neage. At o t h e r W l i t l m s . them i s m m than Mc W Yof a p w r t i m i n g thenutations. FOPexample a t w s i t l m 3 (Table 71, there am three alternative solutions. each r e q u i r i n g t h w e &ationr. F i r s t . t h e r e c w l d be thm T A s u b s t i t u t i m s . m the nt, duck. and ChaChalaCa lincaocs. i n each CIY gemrating a phen)rlalmine t ot y m r i n er e p l a c e m n t Sccmd. there EW. and could be three A - T w b s t l t u t i o n s . on the l i n n g e r leading t o thechicken,the the c o 1 3 n ancestor Of primtes. Thlrd. them Could be me A T change on thechicken lincage. me T * A c h a w on the r a t lineage. and a T A change on the lineage lying bet r c n tk c-n ancestor O f -1s and the c m ancestor Of b i r d s . The three altemat i * solutions a r e t h m averaged and the resulting estimates of nulben of nutations per lineage a t w s i t i o n 3 I R d l f o l l m : cw (0.33). baboon ( 0 ) . human (0) vat(0.67). chicken(0.67). dlck (0.33). chdchdlaca (0.33). c-n ancestor Of p r i m k (0.33). c o m n 4nCeStDr Of m m l s t o cancestor O f b i r d s (0.33). By pmceeding i n t h i s m n n e ~ .we dPPOltianedthe 18 mutations a t the 15 i n f o m t i v e p o s i t i o n s and the 151 mutations a t the 64 u n i n f o m t i w w s i t i m s m the t r e e f o r seven species. The r e s u l t s f a r the m a m a l i m pIrt Of the tm appear i n Fig. 2 ofthe main t e x t .

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