Table Of ContentCTAP-III, (cid:12)TG,
and NAP-2
Ernst Brandt*, Andreas Ludwig and Hans-Dieter Flad
Research Center Borstel, Center for Medicine and Biosciences, D-23845 Borstel, Germany
*corresponding author tel: (cid:135)49-4537-188444, fax: (cid:135)49-4537-188404, e-mail: [email protected]
DOI: 10.1006/rwcy.2000.10005.
SUMMARY aminoacidsequencesonlydifferinthelengthoftheir
N-termini. (cid:12)TG was discovered in 1975 as a product
releasedbythrombin-activatedplatelets(Mooreetal.,
CTAP-III, (cid:12)TG, and NAP-2 are proteolytic trunca-
1975), and its complete primary structure was
tion products derived from a common precursor
reported 3 years later (Begg et al., 1978). The dis-
(PBP) that is synthesized in megakaryocytes and
covery of CTAP-III dates back to 1977, when it was
secretedbyactivatedplatelets.NAP-2isgeneratedby
purified from human platelets and reported to
enzymatic processing by leukocytes and is active on
stimulate hyaluronic acid formation and DNA syn-
neutrophils, affecting chemotaxis, degranulation,
thesis in human connective tissue cells (Castor et al.,
adhesion receptor expression, and respiratory burst
1977). Subsequently CTAP-III was found to be
viahigh-andlow-affinityinteraction withchemokine
identical to a secreted platelet protein termed low-
receptors CXCR2 and CXCR1, respectively. Its pre-
affinity platelet factor 4 (LA-PF4) and to represent a
cursor CTAP-III also affects metabolic activities in
precursor of (cid:12)TG (Niewiarowski et al., 1980).
connectivetissuecellsbyasyetunknownmechanisms.
According to the latter authors, conversion of
In addition to their proinflammatory and reparative
CTAP-III into (cid:12)TG occurs following its release from
functions, these predominantly intravascularly local-
activated platelets and is catalyzed by a platelet-
ized chemokines appear to exhibit proinflammatory
secreted protease. PBP, a highly cationic protein
roles. This is indicated by their modulatory effects,
(pI(cid:24)10) isolated from platelet release supernates in
e.g. on megakaryocytopoiesis, on endothelial prosta-
1980,wasreportedtobemitogenicforSwiss3T3mouse
cyclin secretion, on basophil leukocyte histamine
cells (Paul et al., 1980), but its relationship to CTAP-
release, and even on T cell cytotoxicity.
III and (cid:12)TG became clear only 6 years later, when it
wasrecognizedtorepresentanN-terminallyextended
precursor of the former proteins (Holt et al., 1986).
BACKGROUND
NAP-2, the smallest protein within thisseries, does
not originate directly from platelets, but was dis-
Discovery
covered almost simultaneously in 1989 by two dif-
ferent groups as a cleavage product formed in
TheCXCchemokines,plateletbasicprotein(PBP,94 supernates of platelet-containing peripheral blood
amino acids), connective tissue-activating peptide III leukocyte cultures (Brandt et al., 1989; Walz and
(CTAP-III, 85 amino acids), (cid:12)-thromboglobulin Baggiolini, 1989). Following the discovery of NAP-1
((cid:12)TG, 81 amino acids) and neutrophil-activating (now known as IL-8), NAP-2 represents the second
peptide 2 (NAP-2, 70 amino acids) are homologous chemokine to become recognized as a neutrophil-
and immunologically crossreactive proteins. Their activating mediator, stimulating functions like
1078 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
lysosomal enzyme degranulation (Walz and sheetarrangedinaGreekkeyfold,andbyone(cid:11)helix
Baggiolini, 1989), Fc receptor-dependent phagocyto- at the C-terminus (Figure 1). Oligomer formation,
sis (Brandt et al., 1989), and as shown later, directed dependingontheindividualmolecularspeciesandthe
chemotactic migration (Walz et al., 1989). physicochemical conditions, has been observed.
Alternative names
Main activities and
pathophysiological roles
Due to their immunological crossreactivity with
polyclonal antisera PBP, CTAP-III, (cid:12)TG, and
Both PBP and CTAP-III have been reported to
NAP-2 are also collectively termed (cid:12)TG or (cid:12)TG
exhibit growth factor-like activities on connective
antigen. Becauseofits structuralsimilarity toplatelet
tissuecells,althoughthemitogenicpropertiesofthese
factor4(PF4)and itslower capacitytobindheparin,
proteinsremaincontroversial.CTAP-IIIhashowever
CTAP-III is sometimes referred to as low-affinity
been shown to stimulate various aspects of metabo-
platelet factor 4 (LA-PF4). Initially an alternative
lism in connective tissue cells, including synthesis of
name for NAP-2 was factor C; however, this is no
matrix components and glucose uptake. (cid:12)TG was
longer in use.
found to be devoid of these activities, while NAP-2
was reported to be functional. In contrast with its
precursors PBP, CTAP-III, and (cid:12)TG, which are
Structure
practically inactive in this respect, the main physiol-
ogical role of NAP-2 is that of an activator of
PBP,CTAP-III,(cid:12)TG,andNAP-2aresmallproteins, neutrophil effector functions. These include chemo-
theirmolecularweightsrangingfromabout10kDato taxis, degranulation, upregulation of adhesion mole-
8kDa, depending on the degree of N-terminal cules, and others. However, PBP and CTAP-III may
truncationoftheindividualmolecules.Theseproteins downmodulateNAP-2-inducedfunctions,asspecified
share the basic structural features found in all other below. Potential pathophysiological roles of (cid:12)TG
CXC chemokines, i.e. they contain four cysteine antigen proteins are only beginning to emerge. In
residues atconservedpositions, andthe twocysteines general elevated plasma levels of (cid:12)TG are indicative
most proximal to the N-terminus are separated by of platelet activation and are found in patients with
one amino acid residue, which in (cid:12)TG antigen e.g. peripheral vascular disease, diabetes mellitus and
proteins is a methionine. In all the (cid:12)TG antigens the coronary artery disease. Corresponding findings were
functionally important tripeptide sequence Glu-Leu- reported in patients suffering from rheumatic disease
Arg (ELR motif), located directly upstream from the and renal disease. In these patients CTAP-III depo-
CXC motif is retained. The overall tertiary structure sition in the synovium and kidney, respectively, was
of these molecules corresponds to that of other CXC associated with partial conversion into NAP-2, sug-
chemokines, comprising an extended N-terminal gesting the involvement of these chemokines in path-
loop, followed by three strands of antiparallel (cid:12) ological neutrophil and connective tissue activation.
Figure 1 Basic secondary structure of (cid:12)TG antigen proteins. The most prominent
structuralfeaturesofNAP-2(aminoacidpositions1–70)anditsN-terminallyextended
precursors PBP, CTAP-III, and (cid:12)TG (starting at positions (cid:255)24, (cid:255)15, and (cid:255)11,
respectively)areshown.Thefourconservedcysteineresiduesandthemodeofdisulfide
bridgingareindicatedinblueandthepositionoftheELRmotifishighlightedinred.
The three (cid:12) sheet strands and the C-terminal (cid:11) helix are symbolized by zig-zag lines
andacylinder,respectively(secondarystructureinformationaccordingtoMalkowski
et al., 1995). (Full colour figure can be viewed online.)
CTAP-III, (cid:12)TG, and NAP-2 1079
GENE AND GENE REGULATION Figure 2 Structure and organization of the PBP gene
((cid:12)TG1).Thethreeexonsareshownasboxes,andwithin
the exons the locations of further segments representing
Accession numbers
untranslated regions (light gray), the region coding for
theleadersequence(darkgray),andthosecodingforthe
(cid:12)TG1 (PBP) gene: PIR A39546 (Majumdar et al., mature protein (black) are indicated. Numbers within
1991) these segments stand for the numbers of base pairs.
CTAP-III cDNA: GenBank M11517 (Mullenbach Upstreamfromthecommonlyusedtranscriptionalstart
codon(position1)thereisanalternativesiteatposition
et al., 1986); M54995, M38441 (Wenger et al., 1989);
(cid:255)4. The locations of the regulatory elements (PU box
PIR A37382 (Wenger et al., 1989)
and TATA box) are given in green and blue, respec-
tively. Data are according to Zhang et al. (1997) and
Majumdar et al. (1991), except for the segmentation of
Chromosome location exon1.Thelatterauthors,usinga(cid:12)TGcDNA,assigned
141bp to the leader sequence, while according to
Wenger et al. (1989), using the longer PBP cDNA, the
The gene encoding human PBP, which is the parent
leader sequence comprises only 102bp. (Full colour
molecule of the proteolytic derivatives CTAP-III,
figure can be viewed online.)
(cid:12)TG, and NAP-2, is located on chromosome 4. It
maps to locus 4q12-q13 as a duplicate (termed (cid:12)TG1
and (cid:12)TG2) and occurs in multiple copies (Majumdar
et al., 1991; Wenger et al., 1991). According to one
report (Zhang et al., 1997), the (cid:12)TG2 is a
nonexpressing pseudogene.
Relevant linkages
chemokines, including IL-8, the MGSA/GRO sub-
Besides the PBP genes (cid:12)TG1 and (cid:12)TG2 the
family, and ENA-78, which all contain four exons.
chromosomal region 4q12-q13 contains the genes
Exon 1 consists of an 87bp 50 UTR, followed by
for several other members of the CXC chemokine
102bp coding for the signal peptide. The remaining
family, including those for PF4, IL-8, and MGSA/
46 nucleotides encode the N-terminus of the mature
GRO, GRO(cid:12), GRO(cid:13), but not IP-10 (Wenger et al.,
PBP. Exon 2 is 136bp long and exon 3 contains the
1991; Tunnacliffe et al., 1992). The former genes are
remaining 100bp of the coding region, including the
closely linked to form a clusteron a single700kbSfil
stop codon (TAA). The rest of exon 3 consists of a
fragment. Especially close linkage of the PBP genes
218bp 30 UTR. There are two regulatory sites
exists with that for PF4, which also occurs as a
upstream from the more commonly used transcrip-
duplicate(PF4andPF4alt,respectively).While(cid:12)TG1
tional start site, an alternative one being located at
and PF4 are separated by <7kb, the distance
(cid:255)4bp.AcanonicalTATAboxislocatedat(cid:255)32bpto
between (cid:12)TG2 and PF4alt is approximately 5kb,
(cid:255)25bp (Majumdar et al., 1991). A second regulatory
and within each (cid:12)TG/PF4 duplication the (cid:12)TG-like
element within a pyrimidine-rich tract is located at
gene is upstream of its linked PF4-like gene
(cid:255)78bp to (cid:255)75bp and was found to match a PU box
(Tunnacliffe et al., 1992).
as well as to bind the hematopoietic transcription
factor PU.1, that belongs to the Ets family (Zhang
et al., 1997).
Regulatory sites and corresponding
transcription factors
Cells and tissues that express
the gene
The gene for PBP ((cid:12)TG1) was first cloned by
Majumdar et al. (1991) using a (cid:12)TG cDNA probe
isolated from a cDNA library derived from the Platelets are by far the major source for mature (cid:12)TG
megakaryoblastic cell line DAMI (Majumdar et al., proteins, but exhibit only marginal mRNA expres-
1991). The gene was found to have a total length of sion. They are storage sites for PBP and CTAP-III,
1139bp andtobedivided into threeexons(Figure2). while gene expression and protein synthesis occur
In this respect the gene was similar to that for PF4 during maturation of the precursor cells, the
(three exons), but differed from those for other megakaryocytes. More recently, leukocytes of the
1080 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
myeloid and lymphoid lineage have also been shown 34-residue leadersequence. The mature PBP contains
to express the gene (Table 1). 94 amino acids and constitutes a precursor of further
N-terminally truncated derivatives, the main repre-
sentativesbeingCTAP-III(85aminoacids),(cid:12)TG(81
PROTEIN
amino acids), and NAP-2 (70 amino acids). Further
truncation products have been found, as described
Accession numbers
below. All proteins contain four cysteine residues
(positions 5, 7, 31, and 47) that are conserved in all
PBP: PIR A24448 (Holt et al., 1986) CXCchemokines,andN-terminaltothefirstcysteine
CTAP-III: PIR A93982 (Castor et al., 1983), MIM the ELR-sequence motif, that is present in all CXC
121010, SwissProt P02775 chemokines capable of interacting with chemokine
(cid:12)TG: PIR A90411 (Begg et al., 1978) receptors CXCR1 and CXCR2.
NAP-2: PIR PL0222 (Walz and Baggiolini, 1990)
Description of protein
Sequence
Physicochemical Properties
As shown in Figure 3, PBP is translated as a 128 Like other chemokines, (cid:12)TG proteins are relatively
amino acid precursor protein (pre-PBP), bearing a resistant to denaturing conditions, such as low pH,
Table 1 Human cells and cell lines expressing the PBP gene
Cell type Remarks Reference
Megakaryocyte Major gene-expressing cell type, Evidence derived from
expression during maturation cell lines/platelets (see below)
Platelet Low level of mRNA expression, Wenger et al., 1989;
(probably not regulated) Power et al., 1995
DAMI (megakaryoblastic cell line) Constitutive expression Majumdar et al., 1991
HEL (erythroleukemic cell line) Constitutive expression Zhang et al., 1997
T cells Induction by PHA/PMA Skerka et al., 1993
Monocytes Induction by LPS Iida et al., 1996
Neutrophil granulocytes Induction by LPS Iida et al., 1996
Figure3 Sequencesof(cid:12)TGproteins.Numbersindicatingthepositionofaminoacidsrelatetothemature
NAP-2(residues1–70).Thusthesequenceofpre-PBPstartsatposition(cid:255)58,containingtheleadersequence
(shown on top), the N-terminus of mature PBP (positions (cid:255)24 through (cid:255)14), of CTAP-III (positions (cid:255)15
through(cid:255)12),of(cid:12)TG(positions(cid:255)11through(cid:255)1),andofNAP-2.TherespectiveN-terminalresidueinthe
precursor proteins is shown in blue, the location of the ELR motif is highlighted in red, and the four
conserved cysteine residues are shown in yellow. (Full colour figure can be viewed online.)
ELRC C C C
CTAP-III, (cid:12)TG, and NAP-2 1081
highsalt,andelevatedtemperatures.Thisisprobably tetrameric chemokine (Malkowski et al., 1995), have
due to the presence of two disulfide bridges been reported. The recombinant NAP-2 proteins
connectingCys5toCys31andCys7toCys47,thereby analyzedbytheseauthorswereslightlydifferentfrom
stabilizing the overall conformational structure. the native NAP-2, in that the former (Mayo et al.,
Reducing agents lead to unfolding of the proteins 1994) used a 72-residue molecule bearing an
andlossofbiologicalactivity.Allofthe(cid:12)TGproteins additional Met at the N-terminus and a Tyr at the
show medium affinity for heparin, which may be due C-terminus, while the latter (Malkowski et al., 1995)
to the clustering of positively charged amino acids at useda70-residuemoleculehavingtheMetatposition
certain regions of the molecules, especially at the C- 6 exchanged for Leu. The basic secondary structure
terminus. However, the positive net charge found in data for monomeric and oligomeric NAP-2 are in
most ofthe proteins (See Table 2) doesnot appear to good accordance, consisting of a long N-terminal
represent the only characteristic responsible for open loop, three strands of (cid:12) sheet, followed by an (cid:11)
heparin binding. In contrast to its close relative helix at the C-terminus. However, some minor
PF4,thetendencyof(cid:12)TGproteinstoformoligomers divergence was found in the exact location of these
(dimers and tetramers) is low and decreases with the structural elements as well as of additional ones
extent of N-terminal truncation (Yang et al., 1994). residing within the N-terminus and the connecting
At physiological concentrations (nanomolar) NAP-2 loops (Figure 4). According to Mayo et al. (1994) (cid:12)
generally exists in a monomeric state, while the N- strands 1, 2, and 3 encompass residues K21–K27,
terminally extended precursors, which are released at V34–I40, and R44–D49, respectively, these elements
concentrations in the micromolar range show some are allocated by Malkowski et al. (1995) to residues
dimer formation (Table 2). I19–G26, V34–T39, and R44–L48, respectively. The
C-terminal (cid:11) helix is reported to contain 13 residues
Secondary Structure
(R54–D66) by the former authors, while it is 12
The solution structure of monomeric NAP-2 (Mayo residues long (I55–D66) according to the latter
et al., 1994), as well as the crystal structure of the authors. However, both groups find that the ultimate
Table 2 Physicochemical properties of (cid:12)TG proteins
Property Protein
PBP CTAP-III (cid:12)TG NAP-2
Chain length (amino acids) 94 85 81 70
Molecular weight (Da) 10,262 9288 8861 7624
Isoelectric point (pI) (cid:24)10 (cid:24)8 (cid:24)7 (cid:24)8.8
Number of charged residues 17/13 14/13 13/13 12/9
(cationic/anionic)
Monomer/dimer ratio at
nanomolar concentrations (cid:24)2000/1 (cid:24)500,000/1
micromolar concentrations (cid:24)1000/1 (cid:24)5000/1
Figure 4 Secondary structure of NAP-2. An allocation of secondary structural elements to the primary
sequence of NAP-2 as reported by Mayo et al. (1994) (above) and Malkowski et al. (1995) (below) is
shown.Stretchesofsequencethatareorganizedinto (cid:12) strandsand(cid:11) helicesarehighlightedingreenand
yellow,respectively,whileturnswithintheN-terminusandtheconnectingloopsareboxedinred.Amino
acidsshowninbluerepresentresiduesthatwereintroduced(above)orreplaced(below)intherecombinant
proteins for experimental reasons. (Full colour figure can be viewed online.)
1082 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
fourresidues ofNAP-2 (E67–D70) are notintegrated Figure5 TertiaryandquarternarystructureofNAP-2.A
into the helix, but form fraying ends of undefined schematicoverviewofthetopologyofaNAP-2A/B-dimer.
structure. Single or double hydrogen bonds are indicated by single-
and double-headed arrows (going from the donor to the
acceptor), respectively. Residues within (cid:12) strands 1, that
Tertiary Structure and Subunit Association
contribute hydrogen bonds to the dimer interface are
The tertiary structure of NAP-2 as derived from its circledinred.Theresiduesthatformhydrophobicclusters
four-subunit crystal structure described by in one monomer, and those that contribute to stabilizing
thedimerarehighlightedinyellowandgreen,respectively.
Malkowski et al. (1995) is essentially consistent with
Forreasonsofclarity,onlythecorrespondingresiduesfrom
that of the monomeric chemokine in solution des-
one of the monomers are shown. (Full colour figure can
cribed by Mayo et al. (1994). Both groups agree on
be viewed online.)
the presence of a long N-terminal loop, a triple-
stranded antiparallel (cid:12) sheet arranged in a ‘Greek
key’ with (cid:12) strand 2 hydrogen-bonded to strands 1
and3.TheN-terminalopenloopcontainingtheELR
sequence motif is stabilized by the two disulfide
bridges and by two hydrogen bonds running from
T11 to C47 and D49, respectively. Additionally the
hairpin loop connecting (cid:12) strands 1 and 2 contains a
turn that is folded proximal to the ELR motif. The (cid:12)
strands 2 and 3 are also connected by a hydrogen-
bondedhairpinloop,while aloopcontainingasingle
turn leads into the C-terminal (cid:11) helix that runs
diagonally across the top of the (cid:12) sheet. As derived
from the crystal structure of the tetrameric molecule,
the latter is made up of two pairs of dimers (A/B,
C/D) eachformingan extended(cid:12)-pleatedsheetofsix
strands,whichare arrangedbacktobacktoform the
tetramer. The forces stabilizing the association of
monomers into dimers (e.g. for dimer A/B) are
contributed mainly by hydrogen bonding of residues
Q20 and G26 , L22 and V24 , V24 and L22 , as
A B A B A B
well as G26 and Q20 , which are located within the
A B
(cid:12) strands 1 of the opposing subunits. Further dimer
of the naturally occurring residue Met at position 6
stabilizationiscontributedbyhydrophobicclustering
for Leu in the recombinant NAP-2 used in these
of several residues located within the two (cid:11) helices
studies. The related chemokine PF4, having a Leu at
thatextendoverthe(cid:12) sheetoftherespectiveadjacent
the homologous position, readily forms tetramers.
subunit.WithinthesamesubunitI55 andI58 from
A A In functional terms, neither tetramers nor dimers are
the (cid:11) helix make contact with, e.g. I14 and P16
A A required for NAP-2 biological activity (Rajarathnam
residingintheloopconnecting(cid:12) strands1and2.The
et al., 1997). The reduced ability of NAP-2 to
helix–sheet interface of the AB-dimer is further
oligomerize may also be responsible for its lower
stabilized by contacts between helix residues V59 ,
A heparin binding affinity as compared with PF4.
K62 , and L63 , and (cid:12) sheet residues L22 , V24 ,
A A B B Although the crystal structures of the N-terminally
and V36 (Figure 5).
B extended precursors PBP, CTAP-III, and (cid:12)TG have
not been reported, analyses of a NAP-2 molecule
extended by five N-terminal residues (i.e. CTAP-III
Discussion of crystal structure
[des10];Malkowskietal.,1997)suggestthattheover-
all secondary tertiary structure of the NAP-2 portion
As already mentioned, the tendency of NAP-2 to in these molecules is notchanged. Nevertheless, these
form tetramers and dimers under physiological con- precursor molecules have a higher potential for
ditions is extremely low. In all likelihood the tetra- oligomerization and are not neutrophil stimuli. Both
meric crystal structure described by Malkowski et al. phenomena may be related, since it was found that
(1995) results from the experimental conditions re- the N-terminal extension in CTAP-III [des 10] is not
quired for growing crystals (high protein concentra- disordered,butformsauniquestructureascompared
tion, pH 4.6)and mayalso be duetothe replacement with equivalent residues in other chemokines. By
CTAP-III, (cid:12)TG, and NAP-2 1083
foldingbacktointeractwiththeELRmotif,atypeII similarity exists to MGSA/GRO (41 identical amino
turn is formed by residues L2 through E2, giving rise acids, homology: 59%) and IL-8 (33 identical amino
tofurtherstabilizinginteractions(saltbridges)between acids, homology: 47%). The closest relative among
residue E2 and two arginines (R4/R44) within the nonELR chemokines is the PF4 (37 identical amino
same subunit. This folding leads to masking of the acids,homology:53%),whileotherchemokinesfrom
ELR motif and may therefore be responsible for lack this group (IP-10 and MIG) are only 29%
of receptor binding of N-terminally extended pre- homologous. The observation that synthetic variants
cursors. It may also have implications for oligomer- of PF4, but not IP-10 or MCP-1 modified by the
ization, as it helps to establish novel intersubunit insertion of an ELR motif, acquire the capacity to
interactions, e.g. H-bonds between L3 –K45 and interact with chemokine receptors CXCR1 and
B D
L3 –K45 . These interactions also help to immobi- CXCR2 (Clark-Lewis et al., 1993), highlights the
D B
lize the ELR region. close relationship of PF4 to chemokines of the ELR
subfamily.
There is no murine equivalent for the (cid:12)TG
Important homologies
proteins. The only protein cloned from other species
and identified as a homolog of PBP (61% sequence
TheNAP-2portionof(cid:12)TGproteinsdisplaysrelevant similarity)derivesfromporcineplatelets(Poweretal.,
structural homology to other CXC chemokines of 1995) (Figure 7). Although its N-terminus does not
both the ELR and non-ELR subfamily, while the N- contain the cleavage site required for generation of a
terminal extensions upstream from the ELR motif molecule corresponding to human NAP-2, N-ter-
(as found in (cid:12)TG, CTAP-III, and PBP) are unique minally different variants with neutrophil-stimulating
in sequence and spatial arrangement (Figure 6). capacity have been shown to be released by porcine
With respect to ELR chemokines, highest sequence platelets (Yan et al., 1993).
Figure6 ComparisonofNAP-2primaryandsecondarystructuretootherCXCchemokines.Sequencealignmentof
thehumanNAP-2,PF-4,MGSA,andIL-8molecules.Aminoacidresiduesidenticalinallfourchemokinesareboxed
in green, and conservative exchanges are boxed in yellow. The location of (cid:11) helical (underlined) (cid:12) strand (double
lines)secondarystructuralelementsisindicatedforPF4,NAP-2,MGSA,andIL-8accordingtoZhangetal.(1994),
Malkowskietal.(1995),Kimetal.(1994),andCloreetal.(1990),respectively.Thenumberofresiduesidenticalto
those in NAP-2 (id) is shown at the right. (Full colour figure can be viewed online.)
Figure7 SequencealignmentofhumanandporcinePBP.Acomparisonoftheclonedsequencesofhuman
(Wenger et al., 1989) and porcine PBP (Power et al., 1994) is shown. Including the leader sequences, the
proteins are 61.0% homologous (identical amino acid residues are boxed in green). The cleavage sites
correlating to the N-termini of truncated native isoforms are indicated by arrows. The porcine isoforms
L-NAP-2andS-NAP-2donotcorrespondtohumanNAP-2andaredirectlysecretedbyplatelets(Yanetal.,
1993). (Full colour figure can be viewed online.)
1084 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
Posttranslational modifications
(i.e. at least 10% of NAP-2 potency) is only found
in NAP-2 variants bearing N-terminal extensions of
Glycosylation
maximally three amino acid residues.
The (cid:12)TG proteins do not contain consensus se- C-terminal truncation was first observed in NAP-2
quences for N-glycosylation, nor has O-glycosylation isolatedfromculturesupernatesofplatelet-containing
been reported. However, nonenzymatic glycosylation mononuclear blood cells (Brandt et al., 1993). Sub-
of lysyl residues in CTAP-III has been described sequently native variants of NAP-2 lacking four
(Castor et al., 1990). Only one type of monosaccha- (NAP-2[1–66]) and seven amino acids (NAP-2[1–63])
ride, glucose at a moderate ratio (0–4moles/mol of at the C-terminus were isolated and identified (Ehlert
protein) was found, the number of sugar residues et al., 1995, 1998). These variants had higher neutro-
changing with the preparation analyzed. This type of phil-stimulatingpotencyandgreaterreceptor-binding
glycosylation had no apparent influence on the affinity (by about 3-fold and 5-fold, respectively)
biological activities of the chemokine. thanthefull-sizechemokine(NAP-2[1–70]).Analyses
of recombinant C-terminally truncated variants of
NAP-2 revealed that truncation by itself was not re-
Deamination
sponsibleforenhancementofneutrophil-directedbio-
Further microheterogeneityinCTAP-III is causedby logicalactivity,butthatsuccessiveremovalofthethree
deamination of the N-terminal asparagine. This negativelycharged C-terminal amino acids correlated
variant (CTAP-III/Asp1) is commonly present, with a progressive increase in potencies. The abund-
along with authentic CTAP-III (CTAP-III/Asn1) in anceofanativeCTAP-IIImoleculetruncatedbyfour
preparations ofthe chemokinefrom isolated platelets residues suggests that truncated NAP-2 variants may
as well as from leukocyte culture supernates (Brandt derive from correspondingly truncated precursor
et al., 1989; Van Damme et al., 1989; Castor et al., molecules (Ehlert et al., 1998).
1990). Deamination has no impact on CTAP-III
biological activity.
CELLULAR SOURCES AND
Limited Proteolysis
TISSUE EXPRESSION
The posttranslational modification most relevant to
the biological activities of (cid:12)TG proteins consists of
Cellular sources that produce
limited proteolysis of these molecules, at both their
N- and C-termini. Apart from the major isoforms,
See Table 3.
PBP, CTAP-III, (cid:12)TG, and NAP-2, trace amounts of
a variety of other N-terminally truncated variants
have been found, both in unstimulated platelets and
in platelet suspensions co-cultured with blood leuko- Eliciting and inhibitory stimuli,
cytes. Unidentified platelet-derived as well as leuko- including exogenous and
cyte-associated proteases are likely to be involved in
endogenous modulators
the processing of the molecules. While N-terminal
truncation does not appear to affect the connective
tissue-activating properties of (cid:12)TG proteins (Castor The stimuli that induce PBP gene expression during
etal.,1990),itclearlyhasanimpactontheircapacity megakaryocytedevelopmenthavenotbeenidentified.
to activate neutrophil functions. This is most Although it has been found that thrombopoietin, a
strikingly illustrated by CTAP-III, the major (cid:12)TG major cytokine promoting megakaryocyte matura-
protein stored and secreted by platelets, which by tion,increasesthesynthesisofseveralplatelet-specific
itself is inactive on neutrophils, but acquires high (cid:11)-granule proteins including PF4 (Sasaki et al., 1997;
activity upon its cleavage into NAP-2 by monocytes Phillippetal.,1998),acorrespondingupregulationof
(WalzandBaggiolini,1990)andevenmoreefficiently PBP has not been reported. Interestingly, the platelet
whencleavedbyneutrophilsthemselves(Brandtetal., release products PBP/CTAP-III and PF4 themselves
1991; Ha¨rter et al., 1994). In the case of neutrophils act back on megakaryocyte progenitors to inhibit
there is some evidence for plasma membrane- their maturation (Han et al., 1990), thus indirectly
associated cathepsin G being the processing enzyme. decreasing platelet counts and consequently the
While ithasbeenobservedthatthe neutrophil-stimu- availability of PBP/CTAP-III and PF4.
lating capacity of (cid:12)TG proteins increases with the Evidence has recently emerged that apart from
degree of N-terminal truncation, relevant activity megakaryocytes, other blood cells like neutrophils,
CTAP-III, (cid:12)TG, and NAP-2 1085
Table 3 Human cells and cell lines producing (cid:12)TG proteins
Cell type Remarks Reference
Megakaryocytes Synthesis during development Holt et al., 1988
Platelets Release from granules upon stimulation Moore et al., 1975
Megakaryoblastic cell lines
HEL Expression enhanced by DMSO or TPA Tabilio et al., 1984
MEG-01 Secreted in cell culture Tanabe et al., 1991
CMK-7 Enhanced by TPA or in contact Hoshi et al., 1994
with endothelial cells
T cells
Induced by PHA Iida et al., 1996
Monocytes Induced by LPS Iida et al., 1996
Neutrophil granulocytes Induced by LPS Iida et al., 1996
monocytes, and T cells may be induced to produce Because the formation of NAP-2, the major (cid:12)TG
and secrete so far unidentified variants of these protein active on neutrophils, requires proteolytic
chemokines (Iida et al., 1996). However, platelets cleavage of its platelet-secreted precursors PBP/
remain the predominant source for secreted (cid:12)TG CTAP-III by leukocytes, the availability of NAP-2 is
proteins (serum levels: up to about 30mg/mL) with predominantlyregulatedattheprocessinglevel.From
CTAP-III and PBP representing the major isoforms inhibitionstudiesthereisevidencethatacathepsinG-
storedwithinthesecells.Whiletherelativeamountof like serine protease which is constitutively expressed
CTAP-III ranges from 60 to 90% (Brandt et al., on the neutrophil plasma membrane (Brandt et al.,
1991), PBP is present at a mean proportion of about 1991; Ha¨rter et al., 1994), as well as so far undefined
25%, with no (cid:12)TG detectable (Holt et al., 1988). serine protease(s) and thiol protease(s) derived from
Generally stimuli eliciting platelet aggregation and monocytes (Walz and Baggiolini, 1990)are the major
(cid:11)-granule release also induce the secretion of PBP/ enzymesinvolvedinprocessing.Accordingly,purified
CTAP-III. These include thrombin, the arachidonic cathepsin G (Brandt et al., 1991; Car et al., 1991) as
acid metabolite thromboxane (TXA ), different well as chymotrypsin (Car et al., 1991; Cohen et al.,
2
types of conformationally intact collagen (e.g. types 1992), a protease exhibiting similar cleavage specifi-
I, III, and VI which are found in the vessel wall), city, havebeenshowntogenerateactiveNAP-2from
ADP, and PAF. Enhancement of platelet activation its precursors. Major physiological inhibitors of
and granule release may also occur in response to NAP-2 generation reside in plasma and serum
neutrophil-derived cathepsin G (Chignard and (Ha¨rter et al., 1994), while leukocyte-derived elastase
Renesto, 1994). This protease is released following hasthepotentialtodegradethechemokine(Caretal.,
activation of neutrophils by stimulants such asfMLP 1991). Neutrophil-derived serine protease inhibitors
and TNF(cid:11). Cathepsin G activation of platelets is suchasaprotininandchymostatin,aswellassynthetic
comparable to the potent agonist thrombin. inhibitors like sulfonyl fluorides (PMSF, AEBSF),
Moreover,followingitsbindingtoplateletscathepsin and chloromethyl ketones (Z-GLF-CK), block pro-
G cleaves released PBP/CTAP-III into NAP-2 cessing. Some of the monocyte-associated enzymatic
(Cohen et al., 1992). activityisalsosusceptibletoinhibitionbyleupeptin,a
Prostaglandins PGE and PGI are major physiol- thiol protease inhibitor (Walz and Baggiolini, 1990).
1 2
ogicalinhibitorsofsecretion.Thelatterisreleasedby
endothelial cells upon stimulation by thrombin.
Interestingly, (cid:12)TG has been reported to decrease RECEPTOR UTILIZATION
endothelial PGI production, thus facilitating the
2
formation of a locally restricted thrombus at sites of
See Table 4.
injury(Hopeetal.,1979).Pharmacologicalinhibitors
of secretion are phosphodiesterase inhibitors such as
Identification of NAP-2 Receptors
theophylline, activators of adenylyl cyclase such as
hirudine, and synthetic ligands for prostacycline Among (cid:12)TG proteins specific receptors have only
receptors, e.g. iloprost. been identified for NAP-2. Binding studies with the
1086 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
Table 4 Interaction of NAP-2 with chemokine receptors
Receptor CXCR1 CXCR2 DARC ECRF3
Synonyms IL-8RA/IL-8RI IL-8RB/IL-8RII Duffy
Affinity (K ) (cid:24)500nM 1–4nM (cid:24)5nM (cid:24)5nM
d
Expression Neutrophils Neutrophils Erythrocytes Herpesvirus
Monocytes Monocytes Endothelium saimiri
Mast cells Mast cells
Basophils CD8+ T cells
CD8+T cells NK cells
NK cells
Signaling Chemotaxis Chemotaxis None [Ca2+] flux
i
Degranulation Degranulation
[Ca2+] flux [Ca2+] flux
i i
PLD
Respiratory burst
radiolabeled chemokines revealed that NAP-2, but identified. However, to date only CXCR1 and
not its precursors (PBP and CTAP-III) specifically CXCR2havebeenshown tomediateNAP-2-induced
interact with neutrophil receptors that also bind IL-8 cellularresponsessuchaschemotaxis(Loetscheretal.,
and MGSA/GRO. More detailed comparison of the 1994; Ben-Baruch et al., 1997a).
kinetics of competition for binding sites of these
chemokines led to the prediction of the existences of
Structure–Function Relationship
two distinct populations of binding sites on neutro-
phils with characteristic differences in the binding Like with IL-8 and MGSA/GRO, the N-terminal
capacitiesexistingbetweenNAP-2andMGSAonthe Glu-Leu-Arg- (ELR-) sequence motif of NAP-2 has
one hand and IL-8 on the other (Moser et al., 1991; been demonstrated to be an absolute requirement for
Schumacher et al., 1992; Petersen et al., 1994). These specific binding to CXCR1 and CXCR2 (Yan et al.,
binding sites were cloned and identified as the G 1994; Clark-Lewis et al., 1994). In addition to the
i
protein-coupled heptahelical receptors CXCR1 and ELR motif, other regions on NAP-2 also contribute
CXCR2 (previously termed IL-8RA and IL-8RB, to receptor binding. The ultimate C-terminal amino
respectively) (Holmes et al., 1991; Murphy and acids which are not integrated into the (cid:11) helix and
Tiffany,1991).Followingtransfectionintoeukaryotic formfrayingendsappeartodisturbreceptorbinding,
cell lines CXCR2 was shown to bind IL-8, NAP-2, since their removal leads to the enhancement of
and MGSA/GRO with high affinity (K =1–4nM), binding affinity (Ehlert et al., 1995, 1998). However,
d
whereas CXCR1 exhibited comparable high affinity C-terminal truncation including the leucine at posi-
for IL-8 only and considerably lower affinity for tion63decreasesreceptorbinding,indicatingthatthe
NAP-2 and MGSA (K =500nM) (Loetscher et al., L63, which is highly conserved in CXC chemokines,
d
1994; Ahuja and Murphy, 1996; Hoch et al., 1996; represents a functionally important residue.
Murphy, 1997) (see Table 4). These receptors are Several intersubtype receptor chimeras have been
coexpressed in high numbers predominantly on constructedtoelucidatethestructuralbasisforthedif-
neutrophils, but are also detectable on monocytes, ferential affinity of CXCR1 and CXCR2 for NAP-2
NK cells, CD8(cid:135)T cells, mast cells, and basophils by and related chemokines. Switching of the N-terminal
immunofluorescence labeling (Chuntharapai et al., extracellular part between the receptor types demon-
1994; Morohashi et al., 1995; Lippert et al., 1998; strated that the N-terminus of CXCR2 to a large
Ochsenberger et al., 1999). Several other structurally extent dictates that NAP-2 (as well as MGSA/GRO)
related receptors with high affinity for NAP-2 (and isboundwithhighaffinity,whiletheanalogousdomain
related chemokines) namely the Duffy antigen- of CXCR1 does not appear to contain a high affinity
associated receptor for chemokines (DARC) (Neote binding domain(s) for these chemokines (LaRosa
et al., 1993) on erythrocytes and endothelial cells, as et al., 1992; Gayle et al., 1993). Detailed analyses of
well as herpesvirus-encoded CXC chemokine recep- other receptor chimeras led to the discovery that
tors (ECRF3) (Ahuja and Murphy, 1993), have been additionalregionson thesecond extracellularloopof
