Vol. June
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
14. 1991
COMPLEMENTARY
DNA SEQUENCE
OF HUMAN
NEUTROPHIL
ANTIBIOTIC
WITH EXTENSIVE
HOMOLOGY
TO SERINE
Roque P. Almeida,
Maxine Melchior,
David Campanelli,
688-695
AZUROCIDIN,
AN
PROTEASES
Carl Nathan,
and
Joelle E. Gabay* Beatrice
and Samuel A. Seaver Laboratory,
University Received
April
Medical College, 22,
Department
1300 York Avenue,
of Medicine,
Cornell
New York, NY 10021
1991
SUMMARY: Human neutrophils contain in their azurophil granules four antibiotic proteins with extensive homology to serine proteases, collectively termed serprocidins. Azurocidin is the only member of the group that lacks proteolytic activity. Using a monospecific antibody, we isolated from human bone marrow a cDNA encoding the complete azurocidin protein in its mature form, along with an N-terminal 24 residue hydrophobic peptide. The N-terminal third of the mature protein sequence contains a cluster of positively charged amino acid residues, many of which are predicted to be surface exposed. The primary sequence is highly homologous to elastase, proteinase 3, cathepsin G, T-cell granzymes and other serine proteases. However, azurocidin has Gly for Ser and Ser for His substitutions in the catalytic triad. Southern blot analysis of human genomic DNA suggests the existence of a single azurocidin coding sequence. 0 1991Academic Press,Inc.
Azurophil granules, specialized lysosomes of the neutrophil, contain at least 10 proteins implicated in the killing of microorganisms (l-3). Among these are three serine proteases which are additionally involved in degrading connective tissues: cathepsin G, elastase, and proteinase 3 (PR-3)(4-7). These three proteins have collectively been termed serprocidins (8). Recently, we isolated a 29 kDa azurophil granule antibiotic protein, azurocidin, whose NH2-terminal sequence was highly homologous to the serprocidins but which lacked proteolytic activity (7,9). We have used anti-azurocidin antibodies to isolate a human bone marrow cDNA which encodes the entire mature protein (225 residues), along with an N-terminal 24 residue hydrophobic peptide. In this report, we show the nucleotide and deduced amino acid *To whom correspondence should be adressed at Box 57, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. The abbreviations used are: BPI, bactericidal permeability increasing protein; CAP37, cationic antimicrobial protein of 37kDa; CNBr, cyanogen bromide; CTL, cytolytic T lymphocyte; LPS, lipopolysaccharide; PR-3, proteinase 3. 0006-291X/91
$1.50
688
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BIOCHEMICAL
of azurocidin
and the extensive
No.
sequence serprocidins supporting
AND
BIOPHYSICAL
homology
RESEARCH
of this molecule to the
and to human cytolytic T cell (CTL) granzymes. the existence
of a single azurocidin
COMMUNICATIONS
We also present
coding sequence
evidence
in human genomic
DNA. MATERIALS
AND METHODS
cDNA cloning: Azurocidin was purified as described (7,8). Rabbit polyclonal antibodies were raised against the purified protein and their specificity tested as shown (8). These monospecific anti-azlJrOCidin antibodies were used to screen a human bone marrow cDNA expression library in kgtl 1. The cDNA insert from one of the positive clones (121A) was purified, subcloned, and sequenced by the dideoxynucleotide chain termination method using double-stranded plasmid DNA as template (10) with a kit from US Biochemical Corp. (Cleveland, OH) and deoxyadenosine 5’-[ssS] triphosphate. The 234 bp- insert found in clone 121A was cut with the restriction enzyme Hha I to remove the poly A tail and the resulting 82 bp fragment (see Fig. 1) was used to isolate full-length azurocidin cDNA. The 82 bp probe was purified, [snP]-labeled by random priming (11) and used to screen single plaques from the hgtl 1 library by nucleic acid hybridization (12). The filters were washed as described (13), twice for 15 min at room temperature with 6X SSC/O.l% SDS, twice for 15 min at 37 ‘C with 1X SSC/O.l% SDS and once for 30 min at 65 ‘C with 0.1X SSC/O.l% SDS and exposed to Xomat Kodak film at -70 ‘C for 48 h. One of the positive clones obtained (clone 15.2) containing a 905 bp insert was further analyzed. After initial use of universal primers, sequencing of the clone 15.2 cDNA was continued independently on both strands with sequentially constructed 20-25 mer oligonucleotides to obtain sequence in regions overlapping by 20-40 bp. Results were analyzed by DNASIS and PROSIS software (Hitachi America, Ltd., San Bruno, CA). Alignment to other proteases was scored according to Lipman and Pearson (14) with the program FASTP. Structural predictions were obtained as described (15). Southern Hybridization: A genoblot (Clontech, Palo Alto, CA) containing Eco RI, Hind Ill, Barn HI, Pst I, and Bgl II digests of human placental genomic DNA was hybridized with azurocidin cDNA, [szP]-labeled by random priming. Prehybridization (4h in 5X Denhardt’s solution) and hybridization (24h in 50% v/v formamide) were performed at 42’C (high stringency) in 6X SSC/O.l% SDS with 100 pg/ml sonicated salmon sperm DNA. The blot was washed and autoradiographed as outlined above. RESULTS
Isolation contained
of azurocidin
cDNA: The two strongest
207 bp-sequence An 82 bp-fragment
containing
identified
a putative
27 bp-open reading frame followed
a polyadenylation
signal (AATAAA)
The in 3’ by a
(16) and a poly A tail.
derived from the 121A cDNA was used to probe the Agtl 1 library by
nucleic acid hybridization.
Of the 18 positive clones selected,
-900 bp or more, the size predicted (15.2).
clones
an insert of - 200 bp and one of them (clone 121A) was sequenced.
cDNA of this clone comprised
shows
positive
the nucleotide
8 had inserts
to encode full length azurocidin
and the deduced
amino acid sequence
The open reading frame of 747 bp encodes 689
cDNA.
size of Figure 1
of one of these clones
a 249 amino acid
polypeptide,
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No.
2, 1991
-24
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1 CGGCTGACAGTCCTGGCCCTGCTGGCTGGTCTGCTGGCGTCCTCGAGGGCCGGCTCCAGC R LTV LA L LAG L LAS S RAG S
61
-4
CCCCTTTTGGACATCGTTGGCGGCCGGAAGGCGAGGCCCCGCCAGTTCCCGTTCCTGGCC P L L 0 I V G G R K A R P R 0 F P F
COMMUNICATIONS
S
60 -5 120 16
LA
121 TCCATTCAGAATCAAGGCAGGCACTTCTGCGGGGGTGCCCTCATCCATGCCCGCTTCGTG 17SIQNOG RHFOGGALIHARFV
180 36
181
240 56
TGCTTCCAAAGCCAGAACCCTGGGGTTAGCACCGTGGTGCTGGGT fiFOSONPGVSTVVLG
37
241 GCCTATGACCTGAGGCGGCGGGAGAGGCAGTCCCGCCAGACGTTTTCCATCAGCAGCATG 57 AYOLRRREROSRDTFSISSM
300 76
301 AGCGAGAATGGCTACGACCCCCAGCAGAACCTGAACGACCTGATGCTGCTTCAGCTGGAC 77 SENGYDPODNLNmLMLLOLD
360 96
361 CGTGAGGCCAACCTCACCAGCAGCGTGACGATACTGCCACTGCCTCTGCAGAACGCCACG 97 R E A N L T S S V T I L P L P L 0 N
A
T
420 116
421 117
GTGGAAGCCGICACCAGATGCCAGGTGGCCGGCTGGGGGAGCCAGCGCAGTG~GGGGCGT VEAGTROQVAGWGSORSGGR136
480
481 137
CTCTCCCGTTTTCCCAGGTTCGTCAACGTGACTGTGACCCCCGAGGACCAGTGTCGCCCC LSRFPRFVNVTVTPEDOORP156
540
541 157
AACAACGTGTGCACCGGTGTGCTCAICCGCCGCGGTGGCATCTGCAATGGGGACGGGGGC NNVoTGVLTRRGGI0NGDl-fJG176
600
601 177
ACCCCCCTCGTCTGCGAGGGCCTGGCCCACGGCGTGGCCTCCTTTTCCCTGGGGCCCTGT TPLVmEGLAHGVASFSLGPclJ196
660
661 197
GGCCGAGGCCCTGACTTCTTCACCCGAGTGGCGCTCTTCCGAGACTGGATCGATGGTGTT G R G P D F F T R V A L F R D W I D G
721 217
CTCAACAACCCGGGACCGGGGCCAGCCTAGGGGGGCCTGTGACCTCCCATGGAGCCCAGC LNNPGPGPA* *
780
781
CCCCGCCCTCCACACCTCCGGCGCTCCGCACCCACCTCCCACGGCCCCGCCCCTGCCCCC
840
841
GCTCCGGCCAGAGGGGCCTGGCTGTAATAAAGAAGCCGATCTCTCCTCTGAAAAAAAAAA
900
901
AAAAA
905
V
720 216
225
3'
Fiaure 1. Nucleotide and deduced amino acid sequence of azurocidin. The nucleotide sequence is numbered from the first base of the cDNA insert after the EcoRl linker site. The deduced amino acid sequence (single-letter code) is numbered from the first base of the NH2-terminal residue of the mature protein (+l). Regions for which the amino acid sequence have been determined directly are underlined. These include residues l-20, as determined earlier from the intact protein (7), 21-61, as determined in this study from CNBr fragments (8) and 168-191, as determined by Wilde et al (17) from a tryptic peptide. Discrepancies between determined and deduced amino acid sequence are (giving the position, the deduced amino acid, and the assignment from amino acid sequencing): 26, C for S; 34, R for T; 37, M for V. Residues at the active site of serine proteases are boxed. Cysteines are circled. Potential N-linked glycosylation sites are marked by dots. Stop codons are marked with *. The polyadenylation signal is underlined twice. The 82 bp fragment used to isolate the full length azurocidin cDNA lies between nucleotide 721 and 802. These sequence data have been submitted to the EMBL, GenBank, DDBJ Nucleotide Sequence Databases under the accession number X58794 HU AZUR. including an N-terminal latter corresponds
24 residue peptide and a 225 residue
to azurocidin
amino acid sequence
based on the following
for residues
l-37 matches
of the purified protein except in 3 positions peptide from CNBr cleavage and a tryptic fragment
residues;
all of these were
reported
evidence.
the sequence provided
from the NH2 terminus
690
Second,
the sequence
by Wilde et al (17) provided
identical to the predicted
sequence.
The
First, the deduced
(see Fig 1) (7,this study).
of purified azurocidin
residues
mature protein.
a
of 24
another 25
Third, the deduced
Vol. 177, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 3
HYDROPHOBIC
2
1
0
-1 -2 -3
HYDROPHILIC
-4
ci, (i,
6)
(3)
2. Hydropathy plot of azurocidin according to the algorithm of Kyte and Doolittle (34) with a window of n=6. The lower panel indicates distribution of acidic (A) and basic (B) residues. The numbers in parentheses indicate the number of amino acids at the positions marked by the arrows.
Fiaure
amino acid composition
matches
exactly that determined
for purified azurocidin
(not
shown). Features translation
of the predicted
sequence:
The cDNA
initiation codon. The 5’ end of the cDNA encodes
(Fig 2). The 3’ end of the cDNA contains a polyadenylation
for the mature protein.
glycosylation
isoforms
(7,9).
elastase
sites (Asn-X-Ser/Thr)
Eight cysteine
suggests
residues
Mature azurocidin are present
(5 clustered
codons,
stretch as well as
that the clone encompasses
of 225 amino acids with a Mr of 24,261 for the polypeptide N-linked
a hydrophobic
TAG and TGA termination
signal and a poly A tail, suggesting
full length sequence
molecule)
insert lacks the ATG
is predicted
backbone.
in the COOH-terminal disulfide
to consist
Three potential
and may account
that there may be four intramolecular
the
for apparent third of the
bonds, as in
(18).
The mature protein is predicted long stretches
by the Chou-Fasman
of a-helix totalling 1 l%, and 8 O-sheets
in the case of PR-3, the beta-sheets
algorithm
totalling 23%. Remarkably,
are all predominantly
hydrophobic.
and 15 acidic amino acids in the mature protein (Fig 2), azurocidin calculated
pl of 9.2. Thirteen
N-terminal
third of the molecule.
basic and only three acidic residues Regions
to residues
of the molecule
exposed
(20) correspond
residues
include 9 basic and 4 acidic amino acids.
as
With 24 basic
has an overall are clustered
in the
predicted to be surface
5-11, 18-22, 45-48, 59-70, 76-87;
691
(19) to contain 2
these 40
Vol.
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BIOCHEMICAL
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
:
:
RVGAHDPPADVLDE LGRNRGIASVLDE
:
:
:
225 228 218 235 227
I Ia THRSFKLLDDHETPL THKRY
Figure 3. Alignment of the predicted azurocidin sequence with human serine proteases. Gaps were introduced to maximize alignment. Conserved residues are boxed. The His, Asp, Ser residues comprising the catalytic sites are indicated by asterisks. Residues lining the substrate binding pocket of serine proteases (35) are marked by diamonds. The PR-3 sequence is from reference 8, elastase from reference 27, cathepsin G (Cat G) from reference 28, and human lymphocyte protease (HLP) from reference 36.
Figure 3 compares azurocidin sequence with that of human serine proteases aligned to maximize homology. Azurocidin exhibits the highest homology to PR-3 (45%) and elastase (44%). There is also considerable homology (33%) to cathepsin G and human lymphocyte protease (HLP), the human homolog of murine CTL granzyme B. All 8 cysteines in azurocidin align with the cysteine residues of PR-3 and elastase. Of the 3 potential N-linked glycosylation sites in azurocidin, one (AsrUe) aligns with a corresponding site in PR-3 and elastase and another (Asntoo) aligns with PR-3. The his, asp, ser catalytic triad of the serine proteases found in elastase, cathepsin G, and PR-3 is replaced by sew,
aspq
gly175. Although azurocidin is
33% homologous to cathepsin G, it lacks the peptides IIGGR and HPQYNQR which were proposed to contribute to cathepsin G’s antibacterial activity (21,22).
Southern
Hybridization:
Human genomic DNA was restricted with five
enzymes and hybridized with [32P] labeled azurocidin cDNA. At high stringency, a single strongly hybridizing band was observed in four of the digests (EcoR I, Hind III, Barn HI, Bgl II). One faint additional band was detected after digestion with Barn HI. Thus, there appears to be a single human gene for azurocidin. 692
Pst I digestion yielded
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BIOCHEMICAL
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4. Southern blot of human genomic DNA with azurocidin cDNA as a probe. Humanplacental genomic DNA was digestedto completion with Eco RI, Hind III, Barn HI, Pst I, Bgl II (lanes 1-5) and subjectedto electrophoresisin 0.7% agarose.The digestswere transferred to a nylon membrane,hybridized with [32P]-labeledazurocidin cDNA under high stringency and washed as described in Materials and Methods, then autoradiographedwith an intensifying screen for 48 h at -70 ‘C. Fiaure
three hybridizing bands; the azurocidin coding sequence contains one site for Pst I and the azurocidin gene may also contain introns with site(s) for this enzyme ( Fig
4).
DISCUSSION We report nucleotide and deduced amino acid sequence for a human neutrophil azurophil granule protein with antibiotic activity against Gram-negative bacteria, Gram-positive bacteria and fungi (7,8), named azurocidin.
High homology or
identity had recently been proposed between azurocidin and CAP 37, a cationic antimicrobial protein of reported molecular weight 37,000 Da with selective activity against gram-negative bacteria (23,24). This was based on the finding that the 20 N-terminal amino acid of CAP37 were identical with those reported earlier for azurocidin (7,25). However, azurocidin and CAP37 differed in their reported molecular weights, amino acid composition and spectrum of action, precluding any conclusion about their relationship until the complete sequence of each was obtained. Our isolation of azurocidin cDNA and deduction of its amino acid sequence together with a recent report of the complete amino acid sequence of CAP37 (26) establishes the identity of these two molecules. The two sequences correspond exactly, except that the last 3 amino acid residues predicted from the azurocidin DNA were not found in the CAP37 sequence. Southern blot hybridization suggests the existence of a single azurocidin coding sequence. 693
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The azurocidin
cDNA
encodes
peptide that may represent
for an N-terminal
propeptide
RESEARCH
24 residue
This precursor
the positively
charged
in its N-terminal
(ala-glu) described
exposed.
bactericidal-permeability
does not
for elastase, status
(8,27,28).
The half of
Many of these, including eight Arg, are
Interestingly,
increasing
hydrophobic
third a highly basic domain comprising
amino acid residues.
to be surface
COMMUNICATIONS
sequence
G or PR-3; this dipeptide is thought to confer zymogen
ma?ure protein contains predicted
BIOPHYSICAL
a signal sequence.
contain the 2 residue “activation” cathepsin
AND
the deduced
amino acid sequence
of
protein (BPI) with its highly basic N-terminal
domain shows a similar polarization (29). As proposed in the case of BPI, the surface exposure of positive charges may be a functionally important property of antimicrobial proteins,
involved
in binding to target cells and the mechanism
interacts
with the anionic lipopolysaccharide
bacterial
envelope,
and LPS appears
(LPS), a component
unpublished).
terminal domain may promote an electrostatic displays
no homology
from human or bovine neutrophils
reported
(29-32).
cathepsin
Thus, these 4 antibiotic
term serprocidins.
However,
by its lack of proteolytic showed
azurocidin
activity.
amino acid sequence
ser for his substitution
analysis
in the active site.
physiological
It is tempting
common
evolutionary
3 and
a novel family which we
of a tryptic fragment
we have confirmed
of altered specificies
(33).
proteinase
differs from the 3 other members
have given rise to proteases functions
have been
mass, basic pl and broad spectrum
in the catalytic triad (17).
for azurocidin,
antibacterial
primary structures
proteins constitute
Sequence
a gly for ser substitution
additional
molecular
of azurocidin
By obtaining
a deduced
this observation
Evolutionary
and found an
changes
which then can serve
to speculate
of the group
in DNA new
that the serprocidins
have a
origin. ACKNOWLEDGMFNTS
This work was supported
by NIH grants Al-23807
and CA-4521 8.
REFERENCES 1.
2. 3.
Elsbach, P., and Weiss, antimicrobial systems . J.I. Gallin, I.M. Goldstein Ltd., New York. Lehrer, R. I., and Ganz, Spitznagel, J.K. (1990).
J. (1988). Phagocytic cells : oxygen independent In Inflammation: Basic Principles and Clinical Correlates. and R. Synderman, editors) pp.445-470. Raven Press T. (1990). Blood. 76,2169-2181. J. Clin. Invest. 86,1381-1386. 694
N-
with LPS.
it is closely related to elastase,
G by its amino acid sequence,
antibiotic activity.
interaction
whose
activity of this
The basic nature of azurocidin’s
to any of the nonproteolytic
proteins
In contrast,
of the gram-negative
to play a role in the bactericidal
protein ( J. Gabay and D. Golenbock, Azurocidin
of killing. Azurocidin
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4. 5. 6. 7. a. 9. 10. 11.
12. 13. 14. 15. 16. 17. I
a.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
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Weiss, S.J. (1969). N. Engl. J. Med. 320,365376. Baggiolini, M., Bretz, U., Dewald, B., and Feigenson, M.E. (1976). Agents Actions.8,3-11. Kao, R.C., Wehner, N.G., Skubitz, K.M., Gray, B.H., and Hoidal, J.R. (1966). J. Clin.lnvest. 82, 1963-1973. Gabay, J.E., Scott, R., Campanelli, D., Griffith, J., Wilde, C., Marra, M.N., Seeger, M., and Nathan, C.F. (1969) Proc. Natl. Acad. Sci. 86,5610-5614. Campanelli, D., Melchior, M., Fu, Y., Nakata, M., Shuman, H., Nathan, C.F., and Gabay, J.E. (1990). J. Exp. Med. 172,1709-1715. Campanelli, D., Detmers, P., Nathan, C.F., and Gabay, J.E. (1990). J. Clin. Invest. a5,904-915. Chen, E.Y., and Seeburg,P.H. (1965). DNA (NY) 4,165-l 67. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning:A Laboratory Manual. Second edition, vol 3, pp Al -Al 3. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Denhardt, D. (1966). Biochem. Biophys. Res. Commun. 23, 641-646. Jeffreys, A.J. and Flavell, R.J. (1975). Cell 12,429-439. Lipman, D.J., and Pearson, W.R. (1965). Science (Wash. DC). 227,1435-1441. Devereux, J., Haeberi, P., and Smithies, 0. (19 ) Nucleic Acids Res. 12,387-395. Dayhoff, M.O., Barker, W.C., and Hunt, L.T. (1963). Methods Enzymol. 91,524545. Wilde, C.G., Snable, J.L., Griffith, J.E., Scott, R.W. (1990). J. Biol. Chem. 265,2038-2041 . Navia, M.A., McKeever, B.M., Springer, J.P., Lin, T.Y., Williams, H.R., Fluder, E.M., Dorn, C.P., and Hoogsteen, K. (1969). Proc. Natl. Acad. Sci. USA 86,7-l 1. Chou, P.Y., and Fasman, G.D. (1976). Adv. Enzymol. 47,45-148. Emini, E.A., Hughes, J.V., Perlow, D.S., and Boger, J. (1965). J. Virol. 55636639. Bangalore, N., Travis, J., Onunka, V.C., Pohl, J., and Shafer, W. (1990). J. Biol. Chem. 265,13584-l 3588. Shafer, W.M., Pohl, J., Onunka, V.C., Bangalore, N., and Travis, J. (1991) J. Biol. Chem. 266,112-l 16. Shafer, W.M., Martin, L.E., and Spitznagel, J.K. (1964). Infect. Immun. 45,29-35. Shafer, W.M., Martin, L.E., and Spitznagel, J.K.(1986) Infect. Immun. 53,651-655. Pereira, A., Shafer, W.M., Pohl, J., Martin, L.E., and Spitznagel, J.K. (1990) J. Clin. Invest. 65,1466-l 476. Pohl, J., Pereira, A., Martin, N., and Spitznagel, J.K. (1990). FEBS Letters 272,200-204. Takahashi, H., Nukiwa, T., Yoshimura, K., Quick, C., States, D.J., Holmes, M.D., Whang-Peng, J., Knutsen, T., and Crystal, R.G. (1968). J. Biol. Chem. 263, 1473914745. Salvesen, G., Farley, D., Shuman, J., Przybyla, A., Reilly, C., and Travis, J. (1967). Biochemistry. 26,2289-2293. Gray, P., Flaggs, G., Leong, S., Gumina, R., Weiss, J., Ooi, C., and Elsbach, P. (1969). J. Biol. Chem. 264,9505-9509. Frank, R.W., Gennaro, R., Schneider ,K., Przybylski, M., and Romeo, D. (1990). J. Biol. Chem. 265,16671-l&374. Chung, L.P., Keshav, S., and Gordon, S. (1988). Proc. Natl. Acad. Sci. USA. 65,6227-6231. Ganz, T., Selsted, M.E., Szklarek, D., Harwig, S.S.L., Daher, K., Bainton, D.F., and Lehrer, R.I. (1965). J. Clin. Invest. 76,1427-1435. Neurath, H. (1964). Science 224,350-357. Kyte, J., and Doolittle, R.F. (1962). J. Mol. Biol. 157,105-l 32. Kraut, J. (1977) Annu. Rev. Biochem. 46,331-356. Schmid, J., and Weissmann, C. (1987). J. Immunol. 139,250-256. 695
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
14. 1991
COMPLEMENTARY
DNA SEQUENCE
OF HUMAN
NEUTROPHIL
ANTIBIOTIC
WITH EXTENSIVE
HOMOLOGY
TO SERINE
Roque P. Almeida,
Maxine Melchior,
David Campanelli,
688-695
AZUROCIDIN,
AN
PROTEASES
Carl Nathan,
and
Joelle E. Gabay* Beatrice
and Samuel A. Seaver Laboratory,
University Received
April
Medical College, 22,
Department
1300 York Avenue,
of Medicine,
Cornell
New York, NY 10021
1991
SUMMARY: Human neutrophils contain in their azurophil granules four antibiotic proteins with extensive homology to serine proteases, collectively termed serprocidins. Azurocidin is the only member of the group that lacks proteolytic activity. Using a monospecific antibody, we isolated from human bone marrow a cDNA encoding the complete azurocidin protein in its mature form, along with an N-terminal 24 residue hydrophobic peptide. The N-terminal third of the mature protein sequence contains a cluster of positively charged amino acid residues, many of which are predicted to be surface exposed. The primary sequence is highly homologous to elastase, proteinase 3, cathepsin G, T-cell granzymes and other serine proteases. However, azurocidin has Gly for Ser and Ser for His substitutions in the catalytic triad. Southern blot analysis of human genomic DNA suggests the existence of a single azurocidin coding sequence. 0 1991Academic Press,Inc.
Azurophil granules, specialized lysosomes of the neutrophil, contain at least 10 proteins implicated in the killing of microorganisms (l-3). Among these are three serine proteases which are additionally involved in degrading connective tissues: cathepsin G, elastase, and proteinase 3 (PR-3)(4-7). These three proteins have collectively been termed serprocidins (8). Recently, we isolated a 29 kDa azurophil granule antibiotic protein, azurocidin, whose NH2-terminal sequence was highly homologous to the serprocidins but which lacked proteolytic activity (7,9). We have used anti-azurocidin antibodies to isolate a human bone marrow cDNA which encodes the entire mature protein (225 residues), along with an N-terminal 24 residue hydrophobic peptide. In this report, we show the nucleotide and deduced amino acid *To whom correspondence should be adressed at Box 57, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. The abbreviations used are: BPI, bactericidal permeability increasing protein; CAP37, cationic antimicrobial protein of 37kDa; CNBr, cyanogen bromide; CTL, cytolytic T lymphocyte; LPS, lipopolysaccharide; PR-3, proteinase 3. 0006-291X/91
$1.50
688
Vol.
177,
2, 1991
BIOCHEMICAL
of azurocidin
and the extensive
No.
sequence serprocidins supporting
AND
BIOPHYSICAL
homology
RESEARCH
of this molecule to the
and to human cytolytic T cell (CTL) granzymes. the existence
of a single azurocidin
COMMUNICATIONS
We also present
coding sequence
evidence
in human genomic
DNA. MATERIALS
AND METHODS
cDNA cloning: Azurocidin was purified as described (7,8). Rabbit polyclonal antibodies were raised against the purified protein and their specificity tested as shown (8). These monospecific anti-azlJrOCidin antibodies were used to screen a human bone marrow cDNA expression library in kgtl 1. The cDNA insert from one of the positive clones (121A) was purified, subcloned, and sequenced by the dideoxynucleotide chain termination method using double-stranded plasmid DNA as template (10) with a kit from US Biochemical Corp. (Cleveland, OH) and deoxyadenosine 5’-[ssS] triphosphate. The 234 bp- insert found in clone 121A was cut with the restriction enzyme Hha I to remove the poly A tail and the resulting 82 bp fragment (see Fig. 1) was used to isolate full-length azurocidin cDNA. The 82 bp probe was purified, [snP]-labeled by random priming (11) and used to screen single plaques from the hgtl 1 library by nucleic acid hybridization (12). The filters were washed as described (13), twice for 15 min at room temperature with 6X SSC/O.l% SDS, twice for 15 min at 37 ‘C with 1X SSC/O.l% SDS and once for 30 min at 65 ‘C with 0.1X SSC/O.l% SDS and exposed to Xomat Kodak film at -70 ‘C for 48 h. One of the positive clones obtained (clone 15.2) containing a 905 bp insert was further analyzed. After initial use of universal primers, sequencing of the clone 15.2 cDNA was continued independently on both strands with sequentially constructed 20-25 mer oligonucleotides to obtain sequence in regions overlapping by 20-40 bp. Results were analyzed by DNASIS and PROSIS software (Hitachi America, Ltd., San Bruno, CA). Alignment to other proteases was scored according to Lipman and Pearson (14) with the program FASTP. Structural predictions were obtained as described (15). Southern Hybridization: A genoblot (Clontech, Palo Alto, CA) containing Eco RI, Hind Ill, Barn HI, Pst I, and Bgl II digests of human placental genomic DNA was hybridized with azurocidin cDNA, [szP]-labeled by random priming. Prehybridization (4h in 5X Denhardt’s solution) and hybridization (24h in 50% v/v formamide) were performed at 42’C (high stringency) in 6X SSC/O.l% SDS with 100 pg/ml sonicated salmon sperm DNA. The blot was washed and autoradiographed as outlined above. RESULTS
Isolation contained
of azurocidin
cDNA: The two strongest
207 bp-sequence An 82 bp-fragment
containing
identified
a putative
27 bp-open reading frame followed
a polyadenylation
signal (AATAAA)
The in 3’ by a
(16) and a poly A tail.
derived from the 121A cDNA was used to probe the Agtl 1 library by
nucleic acid hybridization.
Of the 18 positive clones selected,
-900 bp or more, the size predicted (15.2).
clones
an insert of - 200 bp and one of them (clone 121A) was sequenced.
cDNA of this clone comprised
shows
positive
the nucleotide
8 had inserts
to encode full length azurocidin
and the deduced
amino acid sequence
The open reading frame of 747 bp encodes 689
cDNA.
size of Figure 1
of one of these clones
a 249 amino acid
polypeptide,
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-24
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1 CGGCTGACAGTCCTGGCCCTGCTGGCTGGTCTGCTGGCGTCCTCGAGGGCCGGCTCCAGC R LTV LA L LAG L LAS S RAG S
61
-4
CCCCTTTTGGACATCGTTGGCGGCCGGAAGGCGAGGCCCCGCCAGTTCCCGTTCCTGGCC P L L 0 I V G G R K A R P R 0 F P F
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S
60 -5 120 16
LA
121 TCCATTCAGAATCAAGGCAGGCACTTCTGCGGGGGTGCCCTCATCCATGCCCGCTTCGTG 17SIQNOG RHFOGGALIHARFV
180 36
181
240 56
TGCTTCCAAAGCCAGAACCCTGGGGTTAGCACCGTGGTGCTGGGT fiFOSONPGVSTVVLG
37
241 GCCTATGACCTGAGGCGGCGGGAGAGGCAGTCCCGCCAGACGTTTTCCATCAGCAGCATG 57 AYOLRRREROSRDTFSISSM
300 76
301 AGCGAGAATGGCTACGACCCCCAGCAGAACCTGAACGACCTGATGCTGCTTCAGCTGGAC 77 SENGYDPODNLNmLMLLOLD
360 96
361 CGTGAGGCCAACCTCACCAGCAGCGTGACGATACTGCCACTGCCTCTGCAGAACGCCACG 97 R E A N L T S S V T I L P L P L 0 N
A
T
420 116
421 117
GTGGAAGCCGICACCAGATGCCAGGTGGCCGGCTGGGGGAGCCAGCGCAGTG~GGGGCGT VEAGTROQVAGWGSORSGGR136
480
481 137
CTCTCCCGTTTTCCCAGGTTCGTCAACGTGACTGTGACCCCCGAGGACCAGTGTCGCCCC LSRFPRFVNVTVTPEDOORP156
540
541 157
AACAACGTGTGCACCGGTGTGCTCAICCGCCGCGGTGGCATCTGCAATGGGGACGGGGGC NNVoTGVLTRRGGI0NGDl-fJG176
600
601 177
ACCCCCCTCGTCTGCGAGGGCCTGGCCCACGGCGTGGCCTCCTTTTCCCTGGGGCCCTGT TPLVmEGLAHGVASFSLGPclJ196
660
661 197
GGCCGAGGCCCTGACTTCTTCACCCGAGTGGCGCTCTTCCGAGACTGGATCGATGGTGTT G R G P D F F T R V A L F R D W I D G
721 217
CTCAACAACCCGGGACCGGGGCCAGCCTAGGGGGGCCTGTGACCTCCCATGGAGCCCAGC LNNPGPGPA* *
780
781
CCCCGCCCTCCACACCTCCGGCGCTCCGCACCCACCTCCCACGGCCCCGCCCCTGCCCCC
840
841
GCTCCGGCCAGAGGGGCCTGGCTGTAATAAAGAAGCCGATCTCTCCTCTGAAAAAAAAAA
900
901
AAAAA
905
V
720 216
225
3'
Fiaure 1. Nucleotide and deduced amino acid sequence of azurocidin. The nucleotide sequence is numbered from the first base of the cDNA insert after the EcoRl linker site. The deduced amino acid sequence (single-letter code) is numbered from the first base of the NH2-terminal residue of the mature protein (+l). Regions for which the amino acid sequence have been determined directly are underlined. These include residues l-20, as determined earlier from the intact protein (7), 21-61, as determined in this study from CNBr fragments (8) and 168-191, as determined by Wilde et al (17) from a tryptic peptide. Discrepancies between determined and deduced amino acid sequence are (giving the position, the deduced amino acid, and the assignment from amino acid sequencing): 26, C for S; 34, R for T; 37, M for V. Residues at the active site of serine proteases are boxed. Cysteines are circled. Potential N-linked glycosylation sites are marked by dots. Stop codons are marked with *. The polyadenylation signal is underlined twice. The 82 bp fragment used to isolate the full length azurocidin cDNA lies between nucleotide 721 and 802. These sequence data have been submitted to the EMBL, GenBank, DDBJ Nucleotide Sequence Databases under the accession number X58794 HU AZUR. including an N-terminal latter corresponds
24 residue peptide and a 225 residue
to azurocidin
amino acid sequence
based on the following
for residues
l-37 matches
of the purified protein except in 3 positions peptide from CNBr cleavage and a tryptic fragment
residues;
all of these were
reported
evidence.
the sequence provided
from the NH2 terminus
690
Second,
the sequence
by Wilde et al (17) provided
identical to the predicted
sequence.
The
First, the deduced
(see Fig 1) (7,this study).
of purified azurocidin
residues
mature protein.
a
of 24
another 25
Third, the deduced
Vol. 177, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 3
HYDROPHOBIC
2
1
0
-1 -2 -3
HYDROPHILIC
-4
ci, (i,
6)
(3)
2. Hydropathy plot of azurocidin according to the algorithm of Kyte and Doolittle (34) with a window of n=6. The lower panel indicates distribution of acidic (A) and basic (B) residues. The numbers in parentheses indicate the number of amino acids at the positions marked by the arrows.
Fiaure
amino acid composition
matches
exactly that determined
for purified azurocidin
(not
shown). Features translation
of the predicted
sequence:
The cDNA
initiation codon. The 5’ end of the cDNA encodes
(Fig 2). The 3’ end of the cDNA contains a polyadenylation
for the mature protein.
glycosylation
isoforms
(7,9).
elastase
sites (Asn-X-Ser/Thr)
Eight cysteine
suggests
residues
Mature azurocidin are present
(5 clustered
codons,
stretch as well as
that the clone encompasses
of 225 amino acids with a Mr of 24,261 for the polypeptide N-linked
a hydrophobic
TAG and TGA termination
signal and a poly A tail, suggesting
full length sequence
molecule)
insert lacks the ATG
is predicted
backbone.
in the COOH-terminal disulfide
to consist
Three potential
and may account
that there may be four intramolecular
the
for apparent third of the
bonds, as in
(18).
The mature protein is predicted long stretches
by the Chou-Fasman
of a-helix totalling 1 l%, and 8 O-sheets
in the case of PR-3, the beta-sheets
algorithm
totalling 23%. Remarkably,
are all predominantly
hydrophobic.
and 15 acidic amino acids in the mature protein (Fig 2), azurocidin calculated
pl of 9.2. Thirteen
N-terminal
third of the molecule.
basic and only three acidic residues Regions
to residues
of the molecule
exposed
(20) correspond
residues
include 9 basic and 4 acidic amino acids.
as
With 24 basic
has an overall are clustered
in the
predicted to be surface
5-11, 18-22, 45-48, 59-70, 76-87;
691
(19) to contain 2
these 40
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BIOCHEMICAL
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
Azurocidin PR-3 Elastase Cat G HLP
:
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
:
:
RVGAHDPPADVLDE LGRNRGIASVLDE
:
:
:
225 228 218 235 227
I Ia THRSFKLLDDHETPL THKRY
Figure 3. Alignment of the predicted azurocidin sequence with human serine proteases. Gaps were introduced to maximize alignment. Conserved residues are boxed. The His, Asp, Ser residues comprising the catalytic sites are indicated by asterisks. Residues lining the substrate binding pocket of serine proteases (35) are marked by diamonds. The PR-3 sequence is from reference 8, elastase from reference 27, cathepsin G (Cat G) from reference 28, and human lymphocyte protease (HLP) from reference 36.
Figure 3 compares azurocidin sequence with that of human serine proteases aligned to maximize homology. Azurocidin exhibits the highest homology to PR-3 (45%) and elastase (44%). There is also considerable homology (33%) to cathepsin G and human lymphocyte protease (HLP), the human homolog of murine CTL granzyme B. All 8 cysteines in azurocidin align with the cysteine residues of PR-3 and elastase. Of the 3 potential N-linked glycosylation sites in azurocidin, one (AsrUe) aligns with a corresponding site in PR-3 and elastase and another (Asntoo) aligns with PR-3. The his, asp, ser catalytic triad of the serine proteases found in elastase, cathepsin G, and PR-3 is replaced by sew,
aspq
gly175. Although azurocidin is
33% homologous to cathepsin G, it lacks the peptides IIGGR and HPQYNQR which were proposed to contribute to cathepsin G’s antibacterial activity (21,22).
Southern
Hybridization:
Human genomic DNA was restricted with five
enzymes and hybridized with [32P] labeled azurocidin cDNA. At high stringency, a single strongly hybridizing band was observed in four of the digests (EcoR I, Hind III, Barn HI, Bgl II). One faint additional band was detected after digestion with Barn HI. Thus, there appears to be a single human gene for azurocidin. 692
Pst I digestion yielded
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4. Southern blot of human genomic DNA with azurocidin cDNA as a probe. Humanplacental genomic DNA was digestedto completion with Eco RI, Hind III, Barn HI, Pst I, Bgl II (lanes 1-5) and subjectedto electrophoresisin 0.7% agarose.The digestswere transferred to a nylon membrane,hybridized with [32P]-labeledazurocidin cDNA under high stringency and washed as described in Materials and Methods, then autoradiographedwith an intensifying screen for 48 h at -70 ‘C. Fiaure
three hybridizing bands; the azurocidin coding sequence contains one site for Pst I and the azurocidin gene may also contain introns with site(s) for this enzyme ( Fig
4).
DISCUSSION We report nucleotide and deduced amino acid sequence for a human neutrophil azurophil granule protein with antibiotic activity against Gram-negative bacteria, Gram-positive bacteria and fungi (7,8), named azurocidin.
High homology or
identity had recently been proposed between azurocidin and CAP 37, a cationic antimicrobial protein of reported molecular weight 37,000 Da with selective activity against gram-negative bacteria (23,24). This was based on the finding that the 20 N-terminal amino acid of CAP37 were identical with those reported earlier for azurocidin (7,25). However, azurocidin and CAP37 differed in their reported molecular weights, amino acid composition and spectrum of action, precluding any conclusion about their relationship until the complete sequence of each was obtained. Our isolation of azurocidin cDNA and deduction of its amino acid sequence together with a recent report of the complete amino acid sequence of CAP37 (26) establishes the identity of these two molecules. The two sequences correspond exactly, except that the last 3 amino acid residues predicted from the azurocidin DNA were not found in the CAP37 sequence. Southern blot hybridization suggests the existence of a single azurocidin coding sequence. 693
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The azurocidin
cDNA
encodes
peptide that may represent
for an N-terminal
propeptide
RESEARCH
24 residue
This precursor
the positively
charged
in its N-terminal
(ala-glu) described
exposed.
bactericidal-permeability
does not
for elastase, status
(8,27,28).
The half of
Many of these, including eight Arg, are
Interestingly,
increasing
hydrophobic
third a highly basic domain comprising
amino acid residues.
to be surface
COMMUNICATIONS
sequence
G or PR-3; this dipeptide is thought to confer zymogen
ma?ure protein contains predicted
BIOPHYSICAL
a signal sequence.
contain the 2 residue “activation” cathepsin
AND
the deduced
amino acid sequence
of
protein (BPI) with its highly basic N-terminal
domain shows a similar polarization (29). As proposed in the case of BPI, the surface exposure of positive charges may be a functionally important property of antimicrobial proteins,
involved
in binding to target cells and the mechanism
interacts
with the anionic lipopolysaccharide
bacterial
envelope,
and LPS appears
(LPS), a component
unpublished).
terminal domain may promote an electrostatic displays
no homology
from human or bovine neutrophils
reported
(29-32).
cathepsin
Thus, these 4 antibiotic
term serprocidins.
However,
by its lack of proteolytic showed
azurocidin
activity.
amino acid sequence
ser for his substitution
analysis
in the active site.
physiological
It is tempting
common
evolutionary
3 and
a novel family which we
of a tryptic fragment
we have confirmed
of altered specificies
(33).
proteinase
differs from the 3 other members
have given rise to proteases functions
have been
mass, basic pl and broad spectrum
in the catalytic triad (17).
for azurocidin,
antibacterial
primary structures
proteins constitute
Sequence
a gly for ser substitution
additional
molecular
of azurocidin
By obtaining
a deduced
this observation
Evolutionary
and found an
changes
which then can serve
to speculate
of the group
in DNA new
that the serprocidins
have a
origin. ACKNOWLEDGMFNTS
This work was supported
by NIH grants Al-23807
and CA-4521 8.
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with LPS.
it is closely related to elastase,
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interaction
whose
activity of this
The basic nature of azurocidin’s
to any of the nonproteolytic
proteins
In contrast,
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