preserved antibacterial activity in a family of heparin-binding grow

19 Pages·2010·1.69 MB·English

by  Arne Egesten

JBC Papers in Press. Published on March 22, 2010 as Manuscript M109.081232
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M109.081232
MIDKINE AND PLEIOTROPHIN HAVE BACTERICIDAL PROPERTIES – PRESERVED
ANTIBACTERIAL ACTIVITY IN A FAMILY OF HEPARIN-BINDING GROWTH
FACTORS DURING EVOLUTION
Sara L. Svensson,1 Mukesh Pasupuleti,2 Björn Walse,4 Martin Malmsten,5 Matthias Mörgelin,3
Camilla Sjögren,6 Anders I. Olin,1, 3 Mattias Collin,3 Artur Schmidtchen,2 Ruth Palmer,6 and
Arne Egesten1
From Sections for Respiratory Medicine & Allergology,1 Dermatology,2 and Infection Medicine,3
Department of Clinical Sciences Lund, Lund University, University Hospital, SE-221 85 Lund;
SARomics AB,4 P.O. Box 724, SE-220 07 Lund; Department of Pharmacy,5 Uppsala University, SE-
751 23 Uppsala; Department of Molecular Biology,6 Umeå University, SE-901 87 Umeå; Sweden.
Running head: Antibacterial activity of midkine and pleiotrophin
Address correspondence to: Arne Egesten, BMC B14, Tornavägen 10, SE-221 84 Lund, Sweden. Tel:
[phone]; Fax: [phone], email: [email protected]
Antibacterial peptides of the innate activities, may have previously unrealized
immune system combat pathogenic important roles as innate antibiotics.
microbes, but often have additional roles in
promoting inflammation and as growth The innate immune system is an ancient
factors during tissue repair. Midkine (MK) part of host defence and antimicrobial peptides
and pleiotrophin (PTN) are the only two (AMPs) are critical to execute its functions.
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members of a family of heparin-binding The ability to produce AMPs is broadly ow
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growth factors. They show restricted distributed among all plants and animals, lo
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expression during embryogenesis and are enhancing survival in an environment where ded
upregulated in neoplasia. In addition, MK pathogenic microbes are a constant threat (1). fro
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shows constitutive and inflammation- Some AMPs are constitutively produced, while h
dependent expression in some non- the expression of others is induced during the ttp://w
transformed tissues of the adult. In the inflammatory response to infection. Despite w
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present study, we show that both MK and considerable diversity among AMPs and .jb
PTN display strong antibacterial activity, increasing evidence that they affect bacteria in c.o
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present at physiological salt concentrations. a multitude of ways, rupture of bacterial b/
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Electron microscopy of bacteria and integrity plays a key role in the antibacterial g
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experiments using artificial lipid bilayers activity of most AMPs (2). est o
suggest that MK and PTN exert their In most cases, AMPs of the innate immune n J
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antibacterial action via a membrane system serve additional purposes. They can act nu
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disruption mechanism. The predicted as growth factors to promote tissue repair (e.g. ry 5
structure of PTN, employing the previously promoting angiogenesis) and they can recruit , 2
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solved MK structure as a template, indicates and activate inflammatory cells (3). This 19
that both molecules consist of two domains, pluripotency is characteristic of important
each containing three anti-parallel β-sheets. AMPs such as the cathelicidins, the
The antibacterial activity was mapped to the antibacterial chemokines, and the defensins (4,
unordered COOH-terminal tails of both 5, 6, 7, 8).
molecules and the last β-sheets of the NH - Midkine (MK) and pleiotrophin (PTN)
2
terminals. Analysis of the highly conserved constitute a structurally distinct, highly
MK and PTN orthologues from the conserved family, of small heparin-binding
amphibian Xenopus laevis and the fish growth factors (9). MK is a 13.4 kDa protein
Danio rerio suggests that they also harbor consisting of 123 amino acids while PTN is a
antibacterial activity in the corresponding 15.4 kDa protein of 136 amino acids (10, 11).
domains. In support of an evolutionary MK was first identified as a gene activated by
conserved function it was found that the retinoic acid in murine carcinoma cells (12,
more distant orthologue, insect Miple2 from 13). Independently, a chicken homologue was
Drosophila melanogaster, also displays purified from basement membranes at the same
strong antibacterial activity. Taken time (14). PTN was originally described as a
together, the findings suggest that MK and factor promoting the outgrowth of neurites and
PTN, in addition to their earlier described as a mitogenic factor for murine fibroblasts
(14, 15). Both MK and PTN show restricted
1
Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
expression during embryogenesis, being both with and without a preceding
mainly expressed in the central nervous reduction/alkylation procedure (DTT and
system, the respiratory system and the iodoacetamide). Analysis was performed
gastrointestinal tract. They are upregulated in using HPLC-MS/MS on a QTOF Ultima
several neoplastic diseases (16). In humans, API (ESI-MS/MS) (Waters, Manchester,
MK is upregulated during inflammation in UK) coupled to a CapLC (Waters). In
some tissues of the adult and can be detected in addition, non-reduced samples were subject
plasma of healthy individuals (17, 18, 19). In to MALDI-TOF MS on a MALDI micro
addition, MK recruits and activates MX (Waters). Injected samples were
neutrophils, suggesting important roles for this trapped on a C18 pre-column (300 um x 5
growth factor during inflammation (20, 21). mm, 5 um, 100 Å; LC-packings/Dionex,
MK and PTN bind and activate several Stockholm, Sweden) and separated on an
receptors, for example the anaplastic Atlantis C18 reversed phase analytical
lymphoma kinase (ALK) receptor, the receptor column (75 um x 150 mm, 3 um, 100 Å)
protein tyrosine phosphatase β /ζ (RPTPβ/ζ), (Waters). All experimentally obtained
N-syndecan, whereas MK can also bind LDL- peptide and peptide conglomerate masses
related protein (LRP) and the α4β1- and α6β1- were compared with the expected masses
integrins. Ligand binding results in diverse obtained after trypsin cleavage, using the
cellular effects including mitogenic responses FindPept, PeptideMass, and the
and differentiation (22). PeptideCutter tools D
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Both MK and PTN are found in many (http://www.expasy.org). When MK and wn
species – from Drosophila to humans. They PTN were digested with trypsin, without loa
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their origin among insects. The existence of retained sulfide bridges were identified http
two genes would appear to be the result of an using ESI-MS/MS and MALDI-TOF MS ://w
early gene duplication (23) MK and PTN have (26, 27). Sequence coverage spanning the ww
a structure with β -sheets, held together by positions of all cysteines, thus directly .jb
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disulfide bridges , a structure also found in one representing the primary amino-acid .org
class of AMP, namely the defensins (24). In sequence of both chemokines, was obtained by/
addition, MK and PTN are highly cationic and from MS/MS of samples only when trypsin gu
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contain heparin-binding sites, a common digestion was performed after DTT and t o
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feature of AMPs (25). iodoacetamide treatment. These Ja
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Taken together, MK and PTN share many experiments therefore confirm that both ua
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functional and structural features with AMPs. MK and PTN had proper tertiary 5
The results presented here clearly show that conformations, i.e. contained the correct set , 20
1
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human MK and PTN have antibacterial of disulfide bonds. On a quantitative basis,
properties and that antibacterial domains are no thiol-groups were detected in MK and
conserved in MK and PTN orthologues. PTN respectively using 5, 5’-dithiobis(2-
nitrobenzoate) (DTNB), further
demonstrating that all cysteines are engaged
Experimental Procedures in disulfide bond s(28). 20-mer peptides
corresponding to various regions of human
Chemicals and reagents- Recombinant
MK, PTN, and corresponding orthologues
human PTN and MK were from PeproTech
were from Sigma (PEP screen®).
(London, United Kingdom). According to
To analyze antibacterial activity of Miple2
the manufacturer, the purity of MK and
protein, residues 21-279 of Miple2 were
PTN was greater than 98% as determined
subcloned into the pGEX-T2 GST fusion
by SDS-PAGE and HPLC analyses.
protein expression vector (details available
Determination of disulfide bonds in human
upon request). The resulting construct, pGEX-
recombinant MK and PTN was performed
T2:Miple2, was confirmed by sequence
essentially as described (26, 27). In short,
analysis. GST-Miple2 fusion protein was
the proteins were digested using trypsin
induced and purified from E.Coli BL21 (DE3)
(1:20 w/w enzyme: substrate ratio) at 37°C
bacterial lysates by standard protocols using
for 16 hours. Digestions were performed
glutathione sepharose beads. GST-Miple2
2
recombinant protein was proteolytically investigate the importance of heparin-
cleaved with thrombin (4U/mL). After binding regions of MK and PTN in
cleavage, the thrombin was removed with p- antibacterial activity, the proteins were
aminobenzamidine agarose. The sample was incubated with low molecular weight
dialyzed overnight with a molecular weight heparin (Mw 5 kDa; Fragmin™; Pfizer,
cut-off of 15 kDa, followed by concentration. Stockholm, Sweden) at a MK/PTN: heparin
On an SDS-PAGE gel stained with ratio of 1:1 for 20 minutes before exposure
Coomassie, Miple2 was the only visible to bacteria in the viable counts assay.
protein. To rule out that contaminating Liposome leakage assay- The liposomes
GST or thrombin contributed to the investigated were anionic (DOPE/DOPG 75/25
antibacterial activity of Miple2, these mol/mol). DOPG (1,2-Dioleoyl-sn-Glycero-3-
proteins were used in the viable count assay Phosphoglycerol, monosodium salt) and DOPE
with Staphylococcus aureus. Neither GST (1,2-dioleoyl-sn-Glycero-3-phoshoetanol-
(10 µM) nor thrombin (4U/mL) showed any amine) were from Avanti Polar Lipids
antibacterial activity. (Alabaster, AL) and of >99% purity. The lipid
Bacterial Strains and Growth Conditions- The mixtures were dissolved in chloroform, after
Streptococcus pyogenes strain AP1 (40/58) of which the solvent was removed by evaporation
M1 serotype was from the World Health under vacuum overnight. Subsequently,
Organization Collaborating Centre for 10 mM Tris buffer, pH 7.4, with or without
Reference and Research on Streptococci, additional 150 mM NaCl, was added together D
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Prague, Czech Republic. Staphylococcus with 0.1 M carboxyfluorescein (CF) (Sigma, St w
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aureus (strain 5120), Escherichia coli (strain Louis, MO). After hydration, the lipid mixture loa
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37.4), and Pseudomonas aeruginosa (strain 27- was subjected to eight freeze-thaw cycles d
1) were obtained from the Division of consisting of freezing in liquid nitrogen and from
Infection Medicine, Department of Clinical heating to 60°C. Unilamellar liposomes of http
Sciences, Lund University, Sweden. All about Ø 140 nm were generated by multiple ://w
bacteria were routinely grown in Todd-Hewitt extrusions through polycarbonate filters (pore ww
(TH; Difco/Becton & Dickinson, Franklin size 100 nm) mounted in a LipoFast mini- .jb
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Lakes, NJ) liquid medium at 37°C and 5% extruder (Avestin, Ottawa, Ontario, Canada) at .org
CViOab2.l e Count Assay- All bacteria were 2su2b°sCe.q uUennttr gapelp efidl trCatFio nws a(sS erpehmadoevxe dG -b5y0 , tGwEo by gu/
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cultivated to mid-logarithmic phase (optical Healthcare, Uppsala, Sweden) at 22°C, with t o
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density at 620 nm was 0.4) in TH medium, Tris buffer (or Tris plus 150 mM NaCl) as Ja
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washed, and diluted (1:1,000) in incubation eluent. CF release from the liposomes was ua
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buffer (10 mM Tris-HCl, containing 5 mM determined by monitoring the emitted 5
glucose; pH 7.4). P. aeruginosa were grown fluorescence at 520 nm from liposome , 20
1
for 18 hours at 37°C. Fifty µl of bacteria (106 dispersions (10 mM lipid in 10 mM Tris with 5 9
colony forming units (cfu)/ml) were incubated mM glucose, pH 7.5, with or without 150 mM
in the absence or presence of peptides at additional NaCl). An absolute leakage scale
various concentrations, (0.003-1µM) for one was obtained by disrupting the liposomes at
hour at 37°C. To quantify antibacterial activity, the end of each experiment by addition of 0.8
serial dilutions of the incubation mixtures were mM Triton X-100 (Sigma), causing 100%
plated on TH agar plates, incubated overnight release and dequenching of CF. A SPEX-
at 37°C, and the number of cfu determined. fluorolog 1650 0.22-m double spectrometer
In order to investigate the activity of MK (SPEX Industries, Edison, NJ) was used for the
and PTN in the presence of salt, the viable liposome leakage assay. Measurements were
counts assay was also performed after addition performed in at least duplicates at 37°C.
of different concentrations of sodium chloride Transmission Electron Microscopy- Bacteria
to the incubation buffer (5-150 mM). The were cultured to mid-logarithmic phase as
assay was also performed in the presence of described above. S. pyogenes and E.coli were
human plasma (pooled citrated plasma from at incubated for 1 hour at 37°C with 0.15 µM
least 12 donors; Department of Transfusion PTN or MK and 0.4 µM PTN or MK,
Medicine, University Hospital, Lund, Sweden) respectively. To verify that the bacteria were
and sodium chloride (150 mM) at plasma killed, part of the sample was plated on TH
concentrations ranging from 1% to 30%. To agar plates, incubated at 37°C overnight, and
3
the cfu counted the following day. The model with the lowest DOPE assessment score
remaining parts of the samples were processed was selected. The final model of full length
for electron microscopy as described (29). In PTN was built in MODELLER using the
short, samples (5µl) were absorbed for 45 selected models for the NH - and COOH-
2
seconds onto the grids followed by two washes terminal domains as templates. A model
with distilled water. The samples were stained structure of full length human MK (accession
for three seconds with 0.75% uranyl formate number P21741) was created in a similar
droplets, followed by staining for an additional manner as PTN (described above). The model
15–20 seconds with 0.75% uranyl formate. The of MK contains amino acid residues 21 to 143.
samples were investigated using a Jeol JEM Putative signal peptides of Miple1 and Miple2
1230 EX electron microscope operated at were determined using neural networks and
60kV accelerating voltage. hidden Markov models at
Phylogenetic Analysis- MK and PTN amino www.cbs.dtu.dk/services/SignalP as described
acid sequences were retrieved from GenBank (37).
at the NCBI site (Supplemental Table I). Each Radial Diffusion Assay- Radial diffusion assay
sequence was analyzed with the Psi-Blast (RDA) was performed essentially as described
(NCBI) (30) to find orthologue and paralogue earlier (38). E. coli was grown to mid-
sequences. Sequences showing structural logarithmic phase (optical density at 620 nm
homologies of more than 70% were selected. was 0.4) in 10 mL of full strength (3%, w/v)
These sequences were aligned using ClustalW trypticase soy broth (TSB) (BD Biosciences, D
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(31) with Blosum 80 protein weight matrix San Jose, Cal.). The bacteria were washed once w
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settings (32). Internal adjustments were made, with 10 mM Tris-HCl (containing 5mM loa
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taking the structural alignment into account, glucose; pH 7.4). 4x106 cfu were added to 5 ed
utilizing the ClustalW interface mL of the underlay gel (0.03% TSB, 1% low from
(http://bioweb.pasteur.fr/seqanal/interfaces/clu electro endosmosis-type agarose (Low-EEO; http
stalw.html). The level of consistency of each Sigma), 0.02% Tween-20 (Sigma) in 10 mM ://w
position within the alignment was estimated by Tris-HCl) and poured onto a Ø 90 mm Petri ww
alignment evaluating software Tcoffee dish. After agarose solidification, Ø 4 mm .jb
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(www.igs.cnrsmrs.fr/Tcoffee/tcoffee_cgi/index wells were punched and 6 µl of buffer alone or .org
.cgi) (33). MK and PTN amino acid sequences peptide (100µM) were added to each well. b/
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were used to construct a phylogenetic tree Plates were incubated at 37°C for 3h to allow gu
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applying the neighbor-joining method (34). the peptides to diffuse. 5 mL of the overlay gel t o
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The generated tree was rooted with human (6% TSB, 1% low-EEO agarose in 10 mM J
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PTN, and the reliability of each branch was Tris-HCl) was used to cover the underlay gel. ua
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assessed using 1,000 bootstrap replications. Antimicrobial activity was seen as a clearing 5
Molecular modeling- A model structure of full zone around each well after incubating 18-24h , 20
1
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length human PTN (accession number P21246) at 37°C. The assay was performed in triplicates
was created by coupling generated with either the recombinant human MK and
comparative homology models of PTN NH - PTN holoprotein or, overlapping peptides,
2
and COOH-terminal domains respectively. The (comprised of 20 amino acids) derived from
theoretical model of PTN containing amino the mature holoprotein sequences of MK and
acid residues 33 to 96 (NH -terminal domain) PTN (accession numbers NP_001012333 and
2
and residues 97-168 (COOH-terminal domain) CAA37121). Similar overlapping peptides
was built using the NMR solution structures of from the MK and PTN orthologues from
human MK with Protein Data Bank codes Xenopus laevis (accession number P48530;
1MKN and 1MKC, respectively as templates P48533) Danio rerio (accession numbers
(35). The sequence of PTN was aligned against Midkine-related growth factor A NP_571145;
the sequence of MK using the alignment Midkine-related growth factor B NP_571791;
displayed in Fig. 4A. The modeling was PTN NP_001003981) and Drosophila
performed using the program MODELLER melanogaster (accession number miple1
version 9v7 (36). When modeling the NH - NP_612022; miple2 NP_651996) were also
2
terminal domain of PTN, a restraint defining used. All peptides used were water-soluble.
the disulfide bond between cysteines 47 and 76 The peptides were dissolved in water and no
was added. Ten models for each domain were other solvents were used.
generated using the automodel class and the Calculations- Theoretical pI were computed
4
using EXPASy primary structure analysis tool MK and PTN, model lipid bilayers were
(www.expasy.ch/tools/pi_tool.html). Statistical investigated in liposome leakage experiments.
significance was determined based on the Addition of human recombinant MK and PTN
Student’s t-test for paired observations. caused leakage of fluorescent dye from
micelles even at low concentrations of the
RESULTS proteins. Addition of sodium chloride at 150
mM did not affect the interaction between MK,
Human MK and PTN possess PTN and the lipid bilayers (Figs. 3A & B).
antibacterial activity. MK and PTN were Electron microscopy was used to investigate
investigated for possible antibacterial activity effects on the bacterial morphology and
against a panel of pathogenic bacteria, i.e. the integrity during the killing process. S.
gram-positive bacteria Streptococcus pyogenes pyogenes and E. coli were incubated in either
and Staphylococcus aureus as well as the buffer alone, with MK, or PTN (Fig. 3C). In
gram-negative bacteria, Pseudomonas analogy to the liposome results, bacteria
aeruginosa, and Escherichia coli. The bacteria incubated with MK and PTN, showed
were incubated in buffer alone or with membrane protrusions and leakage of
recombinant MK or PTN at concentrations intracellular content, indicating damage to the
ranging from 0.003 µM to 1 µM followed by a bacterial membranes.
viable count assay. Both MK and PTN showed Homology and structure. A comparison of
high antibacterial activity against gram- full-length human MK and PTN proteins D
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positive bacteria (Figs. 1A & B) and slightly displayed an identity of 45% and a similarity w
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lower activity against gram-negative bacteria of 61% (Fig. 4A). Considering that the loa
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(Figs. 1C & D). cysteines are engaged in disulfide-bonds, pI ed
Antibacterial activity of MK and PTN in of 10.4 and 10.2 were calculated for MK and from
presence of salt and plasma. Physiological PTN respectively. The structures of MK and http
concentrations of sodium chloride impair the PTN have been characterized by nuclear ://w
antimicrobial activity of many AMPs, such as magnetic resonance (NMR) and consist of two ww
defensins (39). Saliva, during fasting domains, each made up of three antiparallel β- .jb
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conditions, contains 5 mM sodium chloride, sheets. However, final structure calculations .org
plasma has a concentration slightly below 150 were only made for MK (35, 41). A b/
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mM, while epithelial surfaces have comparative homology model of PTN was gu
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concentrations in between (40). Therefore, we made using MK as a template (Fig. 4B). The st o
chose to study the antibacterial activity of MK tail portions outside of the domains do not n J
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and PTN in the presence of sodium chloride at seem to form stable structures and thus move nua
concentrations ranging from 5-150 mM. Since freely. The domains are stabilized by five ry 5
S. pyogenes itself is sensitive to salt, S. aureus disulfide bonds that, in the case of MK, , 20
1
and E. coli were investigated. The antibacterial probably provide the resistance to high 9
activity was largely unaffected (Fig 2A,) temperature and acidic conditions (9, 42).
except in the case of S. aureus incubated with The secondary structures of MK and PTN
PTN, where a dose-dependent decrease of the are very similar and it is apparent that the β-
antibacterial activity by 23% was seen at 150 sheets of the N-terminal regions are very
mM sodium chloride (Fig. 2B). The presence coherent (Fig. 4B). The structure of the
of plasma is also known to inhibit the activity COOH-terminal parts is more flexible than the
of AMPs. The influence of human citrated N-terminal, but still similar. Many AMPs
plasma on the killing capacity of MK and PTN have heparin-binding properties and
was therefore investigated using S. aureus and heparin-binding sites of both MK and PTN
E. coli in the viable count assay. Already at have been determined (Fig. 4C) (43, 35). The
low concentrations of plasma, a strong critical heparin-binding site of both MK and
inhibition of both MK- and the PTN-induced PTN is found in the COOH-terminal region.
killing was seen, although less pronounced in However, parts of both the NH - and COOH-
2
the case of E. coli (Figs. 2C & D). terminals are needed to form the complete
MK and PTN have membrane-disrupting binding site (43). The charge distribution is
properties. Many AMPs have a mode of action shown in the same figure (Fig. 4C),
that includes disruption of the bacterial plasma demonstrating that MK and PTN are quite
membrane. To explore if this is the case for similar in this respect, although PTN have
5
more negatively charged residues in the tail Antibacterial domains in orthologues. MK
than MK. and PTN orthologues were subsequently
Antibacterial domains of human MK and investigated for the presence of antibacterial
PTN. The MK and PTN holoproteins are activity to test whether this feature has been
antibacterial (Fig. 1) but some regions within conserved evolutionarily. Peptides of 20 amino
the molecules may be of particular importance. acids, with an overlap of 10 amino acids, from
To determine whether the heparin-binding the different orthologues were synthesized. A
sites are involved in exerting the distribution of antibacterial activity, similar to
antibacterial activity, low molecular weight that observed in human MK and PTN, was
heparin was used to block the heparin- found in the corresponding peptides of the
binding sites in MK and PTN. After orthologues proteins (Fig. 5C, Supplemental
preincubation with heparin at equimolar Fig. 1). High antibacterial activity was seen in
concentrations, both MK and PTN lost most a lysine-rich region including cysteines 4-6
of their antibacterial activity (Fig. 4D) (Peptide 5) and in the COOH-terminal tail
demonstrating crucial importance for the including cysteine 10 (Peptide 12) (Figs. 5B &
heparin-binding sites in the antibacterial C). Synthetic peptides, corresponding to the
activity. To further map regions of the regions of Peptides 5 and 12 of the MK and
proteins that contain antibacterial activity, PTN orthologues were examined to compare
overlapping peptides, 20 amino acids in length, their antibacterial activity using RDA. The
were synthesized. These were used to screen observed antibacterial activity was almost D
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for antibacterial activity in other parts of the equally high, when comparing peptide 5 and w
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proteins by radial diffusion assay with E.coli 12 of human and X. laevis while the loa
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(Supplemental Fig. 1). The regions with the corresponding peptides derived from the fish d
highest antibacterial activity were localized in D. rerio were less active (Fig. 5C). In from
the last β-strand in the NH2-terminal domain addition, antibacterial activity of the human http
(peptides 5 and 6) in the middle β-strand of the peptides was assessed in the presence of ://w
C-terminal domain (peptide 9) coherent with sodium chloride at physiological ww
the heparin-binding site. But activity was also concentrations (50-150 mM). PTN5 (10 µM) .jb
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found in two peptides derived from the was almost unaffected by salt while the .org
COOH-terminal tail (peptides 11 and 12). The activity of MK12 decreased by 35% at 150 b/
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location of the regions with the highest mM sodium chloride. The antibacterial gu
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antibacterial activity of MK and PTN are activity of the other peptides decreased by t o
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shown in Fig. 5A. The peptides displayed a approximately 60% in the presence of 150 J
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similar pattern of activity with S. aureus using mM sodium chloride (Supplemental figures ua
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viable count assay (data not shown). 2A, B & D, E). The antibacterial activity of 5
Alignment and phylogenetic tree. To all peptides was abolished in the presence of , 20
1
9
investigate homologies and possible plasma (1-30% plasma; data not shown).
conservation of antibacterial domains during Using the liposome leakage assay, all
evolution, sequence alignment of MK and PTN peptides (MK12, PTN5, and PTN12) except
orthologues in the amphibian Xenopus laevis the MK5-peptide, exerted strong
and the fish Danio rerio were compared with membrane-disruptive activity also in the
their human counterparts (Fig. 5B; Identities presence of sodium chloride (150 mM)
and similarities are presented in Supplemental (Supplemental figures 2C & F).
TABLE II). As a result of gene duplication, Phylogenetic tree. The most distant
D. rerio has two closely related MK genes, orthologues of MK and PTN are Miple1 and
Midkine-Related Factor A and B (MRGFA Miple2 of Drosophila melanogaster. A
and MRGFB) that are distinct from PTN phylogenetic tree was constructed using
(44). Upon alignment of the mature proteins, representative MK and PTN orthologues of
the ten cysteine residues are perfectly invertebrates and vertebrates, yielding
conserved, suggesting that the overall structure information about a common ancestor (Fig.
is conserved among the species investigated. In 6A). During the evolution of vertebrates, MK
addition, the highly cationic regions, with a and PTN are separated and belong to two
content of lysines and arginines, also show a different clades. The high degree of similarity
high degree of similarity. between the two proteins suggests that they
have originated from the same gene by gene-
6
duplication, but it is not evident which one has bilayer, for example the bacterial plasma
diverged and which one is the original (23). membrane. Another property in common with
Antibacterial activity of Drosophila Miple2. β-defensins is that, at least MK, dimerize in
Investigation of sequence alignments of human solution (46). Oligomerization can provide a
MK and PTN with Miple1 and Miple2 of D. more efficient exposure of antibacterial
melanogaster, highlights conservation of all residues to the target organism, as
cysteine residues (Fig. 6B). To see if the early demonstrated for β-defensins (48). Heparin-
predecessors also display antibacterial activity, binding properties, depending on the presence
viable count assays using recombinant Miple2 of specifically arranged lysines in Cardin-
against E.coli and S. aureus, were performed. Weintraub motifs, is a common theme among
These assays clearly demonstrate antibacterial antibacterial peptides (25). In the case of MK
activity of Miple2 against E.coli bacteria (Fig. and PTN, the mapping experiments suggest
6C) but not against S. aureus at the same that the heparin-binding region of the
concentration range (data not shown). Thus, molecules has a high antibacterial activity. In
antibacterial activity of the MK/PTN family of this context, it should also be noted that the
heparin-binding growth factors is clearly overlapping peptides obviously do not exactly
evolutionarily preserved. reflect the complete 3D structure and activity
of the holoprotein. Indeed, as seen in the
molecular models (Fig. 4C), MK and PTN
DISCUSSION both display an amphipathic character with D
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distinct and separated cationic and n
This study shows that the heparin-binding lo
hydrophobic "faces", features typical of most ad
growth factors MK and PTN exert strong e
antibacterial activity against a panel of gram- AMPs. Taken together, the positioning of d fro
cationic amino acid residues is likely to m
positive and gram-negative bacteria, by h
disrupting the integrity of their plasma pArMomPos,t ei ntchleu dminemg bMraKn ea-nddi sPruTpNti.n g properties of ttp://w
membranes. Interestingly, the activity was w
Many AMPs can both act as growth factors w
largely unaffected by the presence of 150 mM and proinflammatory mediators, one example .jbc
sodium chloride, a concentration equal to that .o
being the β-defensins (49, 7). Another rg
of human plasma. Strong antibacterial activity b/
example is the antibacterial fragment LL-37, y
was mapped to the unordered COOH-terminal g
derived from the human cathelicidin, which ue
tail and to the last β–sheet of the NH -terminal s
2 activates the receptor FPRL-1 on neutrophils t o
of both molecules. We also demonstrate that n
and promotes angiogenesis (4, 5). Conversely, Ja
the antibacterial activity is conserved in nu
growth factors, such as HB-EGF, possess a
corresponding regions of MK and PTN ry
antibacterial properties, further illustrating this 5
orthologues, and that the holoprotein of the , 2
most distant orthologue - Drosophila Miple2 multifunctionality (50). MK has neutrophil- 019
activating properties, although the receptor on
exerts strong antibacterial activity.
neutrophils has not been characterized (8).
MK and PTN both contain two domains
Antibacterial chemokines constitute another
consisting of three anti-parallel β-sheets that
family of proteins that both structurally and
are held together by a hinge-region. The β-
functionally resemble MK and PTN.
defensins are an important family of AMPs
Generally, these molecules consist of an NH -
that share structural similarities with MK and 2
terminal region with three anti-parallel β -
PTN. The β-defensins are cationic and have
sheets and a COOH-terminal amphiphatic α-
three anti-parallel β-sheets that are stabilized
helix (51). For example, the CC chemokine
by disulfide-bonds between six cysteine
CCL20 and the CXC chemokine CXCL6, both
residues, similarly to the NH -terminal domain
2 have mitogenic and leukocyte activating
of MK and PTN (45, 46). Additionally, the β-
properties, in addition of being antibacterial
defensins have a short COOH-terminal
themselves (52, 53, 54, 55).
amphiphatic α -helix, containing antibacterial
Looking at the amino acid sequences, MK
activity, which does not seem to be the case for
and PTN are highly conserved among
neither MK nor PTN (47). Instead, the latter
vertebrates. The conserved positions of
molecules have an unordered COOH-terminal
cysteines suggest that the structures of these
with a high content of lysines that may attain a
molecules are very similar among vertebrates.
α-helical structure when inserted into a lipid
Not surprisingly, mapping of the antimicrobial
7
domains in orthologues of higher species (X. Keratinocytes of the human epidermis
laevis and D. rerio) showed the same pattern, constitutively express MK and it is possible
with the highest antibacterial activities in the that both MK and PTN accumulate in the
last strand of the NH -terminal β-sheet and in limited interstitial volume present between
2
the unordered COOH-terminal regions. The cells, where high and antibacterial
COOH-terminal region is also the most concentrations may be reached (9). In
conserved region amongst the orthologues. addition, the heparin-binding properties of
Moreover, the antibacterial feature clearly MK and PTN can provide an immobilized
exists in Miple2, an early MK/PTN orthologue antibacterial gradient on the surface of
of Drosophila, suggesting that the antibacterial epithelial cells.
properties of this family of molecules are A few inflammatory conditions have been
ancient and highly conserved. Intriguingly, investigated for MK expression in mice and
Miple2 is strongly expressed in the endodermal humans. In humans, MK is present in plasma
epithelium of the Drosophila gut, in agreement and increase during inflammatory bowel
with a role as a potential AMP in vivo (23). disease (17, 18, 19). An increased production
In D. rerio the function of MK differs from of MK is also seen in meningitis where
the function in humans, suggesting that there monocytes and other leukocytes may
has been a functional diversification after a contribute to the accumulation of MK at sites
gene duplication resulting in two MK genes of inflammation (18).
(44). In adult humans, MK expression is found MK may play key roles in the pathogenesis D
o
in the small intestine, colon, kidney, and lung of rheumatoid arthritis where increased levels w
n
(56). Whilst in the adult D. rerio, it is only are present in both serum and synovial fluid of loa
d
e
found in the brain (44). In X. laevis, the patients suffering from the disease. In an d
expression (liver, kidney and brain) is more arthritis model, using MK knockout mice, mice from
similar to humans (57). seldom developed disease and these animals http
have a decreased recruitment of leukocytes to ://w
the inflammatory site (21). Thus, MK has ww
inflammation responsive elements in its .jb
c
promoter-region, including NFk-B, explaining .org
expression at sites of inflammation (58). b/
y
In conclusion, these findings suggest that gu
e
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MK and PTN, in addition to their previously t o
n
described activities, can serve important roles J
a
n
as innate antibiotics. Future investigation ua
ry
should reveal whether these properties of MK 5
and PTN are functionally relevant in vivo. , 20
1
9

8
FOOTNOTES
We would like to thank Pia Andersson, Maria Baumgarten, and Lise-Britt Wahlberg for excellent
technical assistance and Fredrik Hugosson for providing recombinant Miple2. The work was
supported by grants from the Swedish Research Council (project 20674), the Swedish Heart and Lung
Foundation (project 20080246), the Medical Faculty at Lund University, Swedish Government Funds
for Clinical Research (ALF), the Foundations of Bergh, Crafoord, Ihre, Hedberg, Kock, Marianne &
Marcus Wallenberg, and Österlund.

The abbreviations used are: MK, Midkine; PTN, Pleiotrophin; TH, Todd-Hewitt broth; cfu, colony
forming units; CF, carboxyfluorescein; BSA, bovine serum albumin.

FIGURE LEGENDS
Fig. 1. Antibacterial activity of human midkine and pleiotrophin. A - D, Midkine (MK) and
pleiotrophin (PTN) were investigated for antibacterial activity using a viable count assay. The gram-
positive pathogens Streptococcus pyogenes (S. pyogenes) and Staphylococcus aureus (S. aureus), and
the gram-negative pathogens Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa),
were grown to mid-logarithmic phase followed by incubation with proteins at the indicated
D
concentrations or in buffer alone, for one hour at 37°C. Serial dilutions were made and after culture o
w
overnight on agar plates the number of colony forming units (cfu) were counted. The number of nlo
a
colonies remaining after exposure to MK and PTN respectively was compared with the number of cfu d
e
d
obtained after incubation in buffer alone, to calculate % killing. The data shown represent mean, fro
±standard deviation from three separate experiments. m
h
ttp
Fig. 2. Antibacterial activity of human MK and PTN in the presence of salt and plasma. To ://w
w
investigate if the presence of sodium chloride and plasma proteins interfere with the antibacterial w
.jb
activity of MK and PTN respectively, the viable count assay was used. S. aureus and E. coli were c
.o
grown to mid-logarithmic phase followed by incubation with MK and PTN, respectively (0.5 µM in rg
b/
the case of S. aureus and 1 µM in the case of E. coli) for one hour at 37°C. Serial dilutions were plated y g
u
and the resulting cfu counted. Decrease of antibacterial activity was seen in the case of E. coli for both e
s
MK and PTN at higher concentrations of sodium chloride. Addition of plasma showed a strong t on
J
inhibition of the antibacterial activity. The data shown represents mean, ±standard deviation from an
u
three separate experiments. ary
5, 2
0
Fig. 3. Membrane-disrupting properties of human MK and PTN. A and B, Membrane-disruptive 1
9
effects of MK (A) and PTN (B) were analyzed by measuring release of carboxyfluorescein from
liposomes in the absence or presence of sodium chloride (150 mM). A dose-dependent leakage, which
was not affected by the presence of sodium chloride, was seen. C, Ultrastructural changes of bacterial
morphology after incubation with MK or PTN was investigated using electron microscopy. S.
pyogenes and E. coli were incubated in buffer alone (control), and in the presence of MK or PTN (both
proteins at concentrations of 0.5 µM in the case of S. pyogenes and 1 µM in the case of E. coli),
respectively. The samples were fixed and processed for negative staining. The electron micrographs
show intact bacteria after incubation in buffer (control) while incubation in the presence of MK and
PTN caused bacterial disintegration with membrane blebbing and leakage of intracellular contents.
Bar=0.5 µm

Fig. 4. Alignment of the human MK and PTN sequences, and predicted structure of PTN. A,
Alignment of the MK and PTN sequences show high homology (45%) and similarity (61%). Ten
cysteine residues are present, six in the NH -terminal domain and four in the COOH-terminal domain.
2
B, The structure of MK was used as a template to predict the structure of PTN. Both molecules contain
two domains consisting of three anti-parallel β-sheets, held together by a hinge-region. PTN has a
longer unordered COOH-terminal tail than MK. C, Models of MK and PTN showing the distribution
of positively charged (blue) and negatively charged residues (red). The heparin-binding sites are
9
indicated (circles). D, To investigate the importance of heparin-binding domains in antibacterial
activity of the holoproteins, MK and PTN were preincubated with low molecular weight heparin
prior to incubation with S. aureus using the viable count assay. A strong inhibition of the
antibacterial activity was seen at equimolar concentrations of heparin and MK/PTN. Heparin
alone did not affect the viability of bacteria. The data shown represents mean, ±standard
deviation from three separate experiments. Statistical significance was determined based on the
Student’s t-test for paired observations.

Fig. 5. Antibacterial regions of human MK and PTN compared with orthologues from Xenopus
laevis and Danio rerio. A, The two regions corresponding to the highest antibacterial activity of
human MK and PTN (Supplemental Figure 1) are indicated in the structural models (peptide 5: red in
MK (upper) and PTN (lower); peptide 12: red in MK (upper) and in PTN (lower)). B, Alignment of
human MK and PTN and the corresponding orthologues from Xenopus laevis and Danio rerio. The
location of peptide 5 and 12 are indicated. C, Possible differences in antibacterial activity of peptides 5
and 12 was compared between species using a radial diffusion assay. Peptide 3 derived from MK
and PTN respectively, showed no activity in initial screening and thus served as negative
controls. E. coli was grown to mid-logarithmic phase, dispersed in agarose that was allowed to
solidify on Petri dishes. Wells were punched and buffer alone or peptides (100 µM) were added to
each well. Plates were incubated at 37°C for 3 h to allow the peptides to diffuse. Antibacterial activity
was seen as a clearing zone around each well after incubation for 18-24 hours at 37°C. Baseline value D
o
of a non-active peptide in the RDA is 0 mm. Experiments were done in triplicates and results are w
n
shown as mean ±standard deviation. loa
d
e
d
Fig. 6. Evolution of MK and PTN. A, Phylogenetic tree showing the evolutionary relationship of from
selected MK and PTN orthologues using the neighbour-joining method. Numbers on the branches http
indicate the reliability of each branch using 1,000 boot-strap replications. B, Alignment of human MK ://w
and PTN sequences with Miple1 and Miple2 of Drosophila melanogaster show complete conservation ww
of the ten cysteine residues. C, The antibacterial activity of Miple2 assessed by viable count assay. .jb
c
The number of cfu remaining after exposure to MK and PTN respectively, were compared with the .org
number of cfu obtained after incubation in buffer alone, to calculate % killing. The data shown b/
y
represent mean, ±standard deviation from three separate experiments. gu
e
s
t o
n
J
a
nu
a
ry 5
, 20
1
9

10