of October 22, 2014.
This information is current as
and in the Infant Intestine
Early Life: Diverse Roles in Amniotic Fluid
The Salivary Scavenger and Agglutinin in
Loimaranta, Willem Meindert de Vos and Seppo Meri
Miguel Rodriguez, Esther Jimenez Quintana, Vuokko
Martin Parnov Reichhardt, Hanna Jarva, Mark de Been, Juan
published online 15 October 2014J Immunol
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The Journal of Immunology
The Salivary Scavenger and Agglutinin in Early Life: Diverse
Roles in Amniotic Fluid and in the Infant Intestine
Martin Parnov Reichhardt,*
Mark de Been,
Juan Miguel Rodriguez,
Esther Jimenez Quintana,
Willem Meindert de Vos,*
The salivary scavenger and agglutinin (SALSA), also known as gp340 and dmbt1, is an antimicrobial and inﬂammation-regulating
molecule located at the mucosal surfaces. The present study revealed that SALSA was present in the amniotic ﬂuid (AF) and ex-
ceptionally enriched in both meconium and feces of infants. Based on immunological and mass spectrometric analysis, SALSA was
estimated to constitute up to 4–10% of the total protein amount in meconium, making it one of the most abundant proteins. SALSA
proteins in the AF and intestinal samples were polymorphic and exhibited varying polypeptide compositions. In particular,
a different abundance of peptides corresponding to functionally important structures was found in the AF and intestinal SALSA.
The AF form of SALSA had a more intact structure and contained peptides from the zona pellucida domain, which is involved in
cell differentiation and oligomerization. In contrast, the intestinal SALSA was more enriched with the scavenger receptor cysteine-
rich domains. The AF, but not the meconium SALSA, bound to Streptococcus pyogenes,S. agalactiae,S. gordonii, and Escherichia
coli. Furthermore, differential binding was observed also to known endogenous ligands C1q, mannose-binding lectin, and secre-
tory IgA. Our results have thus identiﬁed mucosal body compartments, where SALSA is particularly abundant, and suggest that
SALSA exhibits varying functions in the different mucosal locations. The high levels of SALSA in AF and the infant intestine
suggest a robust and important function for SALSA during the fetal development and in the mucosal innate immune defense of
infants. The Journal of Immunology, 2014, 193: 000–000.
Innate immunity is extremely important for the mother in the
course of maintaining a healthy pregnancy, as well as for the
infant in the early stages of life. The mucosal surfaces are sites
with a very tight yet dynamic regulation of the immune defense
system (1, 2). Newborns have not yet developed a fully functional
adaptive immune system at the time of birth. Therefore, they rely
greatly on the innate immune system (3). In early life, both the
fetus and newborn experience alternating immune challenges (4).
These include avoidance of a harmful immune response from the
mother, which could lead to preterm birth, protection against in-
fection, and coping with the transition from a mostly sterile in-
trauterine environment into a world that is full of foreign Ag
challenges. The latter becomes particularly important during the
initial colonization of the skin and intestinal tract through the ﬁrst
months of life (5, 6).
The salivary scavenger and agglutinin (SALSA), also known as
gp340, salivary agglutinin, and deleted in malignant brain tumor
1 (dmbt1), is a 340-kDa glycoprotein (GenBank accession number
BAA78577.1) (7, 8). SALSA is expressed by a variety of mucosal
tissues throughout the body. It is associated with the epithelial
layer in the lungs, mouth, trachea, gastrointestinal tract, and va-
gina (9–13). Several soluble forms of SALSA have been found
in body ﬂuids lining the mucosal surfaces such as saliva, lacrimal
ﬂuid, and pancreatic juice (7, 14, 15). Notably, SALSA has not
been observed in human blood or plasma.
Multiple functions have been suggested for SALSA including
roles in epithelial differentiation and innate immunity at the mu-
cosal surfaces (10). The immune functions of SALSA are apparent
through its well-established ability to bind and agglutinate a broad
spectrum of both Gram-negative and Gram-positive bacteria, as
well as viruses (16–22). In several cases, such as with Salmonella
enterica,Streptococcus mutans, HIV-1, and inﬂuenza A virus,
SALSA has been shown to have a direct effect on controlling the
infection (16, 18, 23, 24). SALSA’s antimicrobial effects are
mediated in concert with other innate immune molecules such as
mucin 5B, IgA, and surfactant proteins A and D, all of which act
as endogenous ligands for SALSA (7, 25–27). The complement
components C1q and mannose-binding lectin (MBL) are other
targets for SALSA. Through these interactions, SALSA has been
shown to regulate complement activation on surfaces and in so-
Alongside the role of SALSA in innate immunity, a function in
epithelial and stem cell differentiation has been suggested. Much
*Department of Bacteriology and Immunology, Haartman Institute, University of
Helsinki, Helsinki FI-00014, Finland;
Immunobiology Research Program, Research
Programs Unit, University of Helsinki, Helsinki FI-00014, Finland;
Central Hospital Laboratory, Helsinki FI-00029, Finland;
Department of Veterinary
Biosciences, University of Helsinki, Helsinki FI-00014, Finland;
Department of Medical
Microbiology, University Medical Center Utrecht, Utrecht 2584 CX, the Netherlands;
Department of Nutrition, Food Science and Food Technology, Complutense University
of Madrid, Madrid 28040, Spain;
Department of Medical Biochemistry and Genetics,
University of Turku, Turku FI-20014, Finland; and **Laboratory of Microbiology,
Wageningen University, Wageningen 6708 PB, the Netherlands
Received for publication June 26, 2014. Accepted for publication September 2, 2014.
This work was supported by the Helsinki Biomedical Graduate Program, Helsinki
University Central Hospital funds, the Helsinki University Central Hospital Labora-
tory, the Sigrid Juse
´lius Foundation, Helsinki University Funds, the Stockmann Foun-
dation, and the Academy of Finland.
Address correspondence and reprint requests to Dr. Seppo Meri, Department of
Bacteriology and Immunology, Haartman Institute, P.O. Box 21, University
of Helsinki, Helsinki FI-00014, Finland. E-mail address: seppo.meri@helsinki.ﬁ
Abbreviations used in this article: AF, amniotic ﬂuid; cZP3, zona pellucida domain
from chicken sperm receptor 3; dmbt1, deleted in malignant brain tumor 1; GAS,
group A streptococcus; GBS, group B streptococcus; LC-MS/MS, liquid chroma-
tography-tandem mass spectrometry; MBL, mannose-binding lectin; SALSA, sali-
vary scavenger and agglutinin; SRCR, scavenger receptor cysteine-rich; TBS/Ca,
TBS containing 1 mM Ca
; TBS/Ca/Tween, TBS containing 1 mM Ca
0.05% Tween-20; ZP, zona pellucida.
Copyright Ó2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
Published October 15, 2014, doi:10.4049/jimmunol.1401631
at Terkko National Library of Health Sciences on October 22, 2014http://www.jimmunol.org/Downloaded from
of this evidence comes from work with animal orthologs of
SALSA, such as rabbit hensin and mouse CRP-ductin (29, 30).
Fewer studies have been performed in humans. However, a role in
the epithelial development was suggested based on SALSA’s lo-
calization and increased expression in both fetal skin, lung, and
gut compared with adults (8, 10).
SALSA belongs to the scavenger receptor cysteine-rich (SRCR)
protein family. Its N terminus contains up to 13 SRCR domain
repeats separated by scavenger interspersed domains. These are
followed by two C1r/C1s Uegf Bmp-1 domains surrounding the
14th SRCR domain and ﬁnally a zona pellucida (ZP) domain (9).
The dmbt1 gene undergoes alternative splicing giving rise to
differentially sized mRNAs encoding 8–13 N-terminal SRCR
domains (10, 31). At the protein level, two different isoforms have
been identiﬁed in both saliva and lacrimal ﬂuid (7, 14). One study
has estimated that up to 25% of the molecular mass of SALSA is
made up of carbohydrate. The observed protein polymorphism is
thus a consequence of variation in both the protein core and the
glycosylation patterns (32, 33).
Innate immunity is extremely important for both the mother, in
maintaining a healthy pregnancy and subsequent lactation, as well
as for the infant in the early stages of life. In the current study,
exceptionally high levels of SALSA were found immediately be-
fore birth (in amniotic ﬂuid [AF]) and shortly after birth (in me-
conium and feces after 1 wk). The SALSA proteins in the AF and
intestinal samples were found in multiple forms and with varying
functional activities. Different binding abilities to both microbial
and endogenous ligands were found between SALSA in the fairly
sterile microenvironment surrounding the fetus in the womb and
SALSA in the intestine, when the infant encounters a colonizing
microbiota for the ﬁrst time. Our results thus indicate SALSA as an
important innate defense molecule in early life.
Materials and Methods
AF, meconium, and fecal samples (n= 9 for each) were collected at
the Obstetrics or Neonatology Units of Hospital Universitario Doce de
Octubre (Madrid, Spain). AF was collected before rupture of membranes
either during caesarean section (n= 4) or intravaginally (n= 5) before
delivery. Meconium was collected from term newborns after spontaneous
evacuation within the ﬁrst 2 h from birth and before feeding was started. To
avoid potential bacterial contamination arising from the contact between
meconium and perianal skin/nappies, the outer surface of each meconium
sample was removed using a laser scalpel. Then, an internal meconium
portion was submitted to analysis. The fecal samples were collected di-
rectly into sterile containers 1 wk after birth. All sample types were stored
at 280˚C after collection. All infants were delivered by healthy mothers
after a normal pregnancy and breastfed after birth. All parents provided
written informed consent, and the study was approved (protocol B-06/262)
by the Ethical Committee in Human Clinical Research of Hospital Clinico
San Carlos (Madrid, Spain).
Proteins were mechanically extracted from dried AF or unprocessed me-
conium and fecal material by bead beating as described previously (34).
AF aliquots (50 ml) were dried by speedvac and resuspended into PBS
(pH 7.4) followed by sonication. Thawed meconium or fecal material
(125 mg) was resuspended in 375 ml PBS and sonicated. Then, all samples
were subjected to a FastPrep 24 (MP Biomedicals) according to the man-
ufacturer’s instructions. The resulting protein extracts were stored at 280˚C.
To visualize SALSA samples were diluted diversely in TBS (140 mM NaCl
and 5 mM Tris [pH 7.4]) containing 1 mM Ca
(TBS/Ca) (AF7 and AF8,
1:10; M7, AF9, and M9, 1:20; F7, M8, and F9, 1:50; and F8, 1:100). After
dilution, all samples were mixed with nonreducing SDS-PAGE loading
buffer and loaded (10 ml) onto a 4–12% gradient SDS-PAGE gel (Life
Technologies). The proteins were blotted onto a nitrocellulose membrane
(Life Technologies). Nonspeciﬁc binding to the membrane was blocked
with 5% nonfat dry milk in PBS with 0.05% Tween 20 (PBS/Tween).
Mouse monoclonal anti-SALSA Ab (Hyb 213-06; Bioporto, Gentofte,
Denmark) was added to the milk solution (1:10,000) and incubated over-
night at 4˚C. After washing, a secondary HRP-conjugated rabbit anti-
mouse IgG Ab (Jackson ImmunoResearch Laboratories, West Grove,
PA) was added (1:10,000 in PBS). The bands were visualized by elec-
Glycoprotein and lectin staining
SALSA was puriﬁed from AF as described (17). Puriﬁed SALSA (10 ml)
was loaded in triplicate onto 7.5% SDS-PAGE gel. After electrophoresis,
the gel was cut in three parts. One part was stained for protein with silver
nitrate and the second part for glycoproteins with periodic acid-Schiff
reagent (Glycoprotein Staining Kit; Pierce) according to the manu-
facturer’s instructions. The last part of the gel was blotted on polyvinyl-
idene diﬂuoride membrane (Amersham) for staining with DIG-labeled
sialic acid–speciﬁc Sambus Nigra lectin (DIG Glycan Differentiation Kit;
Boehringer Mannheim). Nonspeciﬁc binding was blocked with 3% BSA
and the membrane incubated with the lectin (1:1000) overnight at 4˚C.
Lectin binding was detected with anti–dig-AP according to the manu-
facturer’s instructions. Bovine submaxillary gland mucin and unrelated
bacterial surface proteins were used as positive and negative controls,
respectively, in the glycoprotein and lectin staining assays.
Quantiﬁcation of SALSA in protein extracts by ELISA
To quantify the concentration of SALSA in the different samples, the
extracts were diluted in TBS/Ca. The diluted samples were coated directly
onto Maxisorp plates (Nunc, Roskilde, Denmark). SALSA puriﬁed from
saliva was used as a protein concentration standard. After coating, the plates
were blocked with 5% nonfat milk in TBS/Ca. The plates were washed with
TBS/Ca containing 0.05% Tween-20 (TBS/Ca/Tween). SALSA levels were
detected using monoclonal anti-SALSA (0.05 mg/ml) and HRP-conjugated
rabbit anti-mouse Abs (1:10,000 in TBS/Ca). OPD tablets (DakoCyto-
mation, Glostrup, Denmark) were used for development, and the color
reaction was measured with an iEMS Reader MF (Labsystems, Espoo,
Finland) at an OD of 492 nm.
Liquid chromatography-tandem mass spectrometry
Protein extracts were separated on a one-dimensional polyacrylamide gel.
By using a prestained marker (Bio-Rad) as an m.w. indicator, each sample
lane on the stained gels was further divided into four regions. For fecal and
meconium samples, these regions roughly ranged from: .250–75 kDa, 75–
50 kDa, 50–30 kDa, and 30 to ,10 kDa. For AF samples, the gel lanes
were cut from .250–70, 70–60, 60–35, and 35 to ,10 kDa. NanoLC and
LTQ-Orbitrap-MS analyses, including quality checks and machine cali-
brations, were performed as described (35). For tandem mass spectrometry
(MS/MS) spectral identiﬁcations, an in-house database was constructed
containing a comprehensive set of protein sequences that can be expected
to occur in the (infant) gastrointestinal tract.
Bacterial binding of SALSA
Group A Streptococcus (GAS; ATCC 19615), group B Streptococcus
(GBS), a clinical blood isolate, isolated and identiﬁed at the Helsinki
University Central Hospital Laboratory, and S. gordonii, DL1 Challis (20),
were grown in Todd-Hewitt media overnight at 37˚C. Escherichia coli
(urine isolate) and Salmonella serovar Typhimurium (fecal isolate) were
grown overnight at 37˚C with shaking in Luria broth. AF, meconium, or
fecal protein extracts were diluted in TBS/Ca to a ﬁnal concentration of 0.5
mg/ml SALSA and incubated with 10
bacterial cells in a 50-ml suspension
for 1 h at 37˚C. After incubation, the bacteria were centrifuged at 10,000 3g.
The supernatants were collected, and the bacteria were washed three
times in TBS/Ca. Bound SALSA was eluted by incubating the bacteria in
50 ml nonreducing SDS-PAGE loading buffer (Life Technologies) con-
taining 10 mM EDTA. Binding was visualized by Western blotting. For
each sample, SALSA in the starting material was compared with SALSA
in the supernatant after absorption with bacteria and SALSA eluted from
the surface of the bacteria. To control that no factors in meconium were
inhibiting the SALSA binding, AF was mixed with varying amounts of
meconium. We thus analyzed whether the eluted amount of SALSA pro-
tein was affected by an increasing amount of meconium.
Endogenous ligand binding of SALSA
Binding of SALSA from AF and meconium was tested to known ligands
of salivary SALSA: secretory IgA (Sigma-Aldrich), C1q (Quidel), and
2 THE SALIVARY SCAVENGER AND AGGLUTININ (SALSA) IN EARLY LIFE
at Terkko National Library of Health Sciences on October 22, 2014http://www.jimmunol.org/Downloaded from
recombinant MBL (28). Each protein (1 mg/ml) was coated onto Maxisorp
plates (Nunc, Roskilde, Denmark). After coating, the plates were blocked
with 1% BSA in TBS/Ca/Tween and then washed with TBS/Ca/Tween. AF
and meconium samples were diluted into a SALSA concentration of 1 mg/ml
and incubated in the wells 1 h at 37˚C. SALSA binding was detected using
mouse monoclonal anti-SALSA (0.05 mg/ml) and HRP-conjugated rabbit
anti-mouse Abs (1:10,000 in TBS/Ca). Binding was measured as described
above for quantiﬁcation of SALSA in protein extracts.
SALSA in AF, meconium, and infant feces
To improve our understanding of the role of SALSA in early life,
we studied its presence in AF, meconium, and feces (1 wk after
birth) samples serially collected from a cohort of nine infants. This
allowed a comparison of biological material from mucosal surfaces
of an individual both within the uterus and in the early days of life.
Proteins were extracted from the AF, meconium, and feces samples.
To characterize the SALSA protein in the samples, they were
analyzed by Western blotting for individual differences in size and
band patterns. Examples from three newborn individuals are dis-
played and compared with SALSA in saliva from a healthy adult in
Bands immunoreactive with SALSAwere found in all samples in
the area of the expected 340 kDa under nonreducing conditions.
However, there was a great variation in the appearance of SALSA,
not only from individual to individual in the same body com-
partment, but also from different compartments within the same
individual. This indicated heterogeneity in size and composition of
the proteins. In AF, SALSA appeared either as one thick band or as
a smeared double band (case 7). Cases 8 and 9 seemed to have only
the higher m.w. thick band. The meconium SALSA bands pre-
sented as much wider smears on the blot compared with SALSA
in both AF and fecal samples. Within the smears, some individual
bands were observed. For example, in case 7, meconium SALSA
appeared to have a double band with the higher one at ∼200 kDa
and the lower one at ∼150 kDa. For both case 7 and 9, the me-
conium SALSA smear clearly appeared at a lower m.w. area than
AF SALSA. In case 8, SALSA in meconium appeared as a higher
m.w. band than in AF. Yet, there was a wide smear extending from
the high m.w. area to ∼150 kDa. Finally, the bands observed in the
fecal samples appeared as more distinct bands, similar to those in
the AF samples.
A positive reaction was observed, when AF samples were stained
with periodic acid-Schiff and the sialic acid–speciﬁc Sambucus
nigra lectin. This indicated that SALSA is glycosylated and
contains carbohydrates with terminal sialic acids (data not shown).
The heterogeneity in size and appearance of the SALSA bands
suggested both structural differences among the proteins detected
in the different compartments as well as variations in glycosyla-
tion or other posttranslational modiﬁcations.
To determine the levels of SALSA in the different compartments
during early life, the protein extracts were analyzed by ELISA or
digested with trypsin and subjected to liquid chromatography-
MS/MS (LC-MS/MS). As expected, we were able to identify and
semiquantify peptides that matched to the SALSA protein in all 27
samples and thus in all 3 sample types. The LC-MS/MS analysis
was used to compare the relative abundances of SALSA based on
the number of spectra matching to SALSA-speciﬁc peptides in
relation to the total amount of protein spectra in a given sample
(Fig. 2A). The relative abundance of SALSA was 0.53% for AF,
4.16% for meconium, and 2.81% for feces. Thus, the levels of
SALSA were signiﬁcantly higher in both meconium and feces
compared with AF (p= 0.002). The ELISA analysis provided actual
protein concentration measurements in the protein extracts (Fig. 2B).
The average values were 2.1 mg/ml (range 0–11.5 mg/ml) for AF,
45.8 mg/ml (range 2.8–294.6 mg/ml) for meconium, and 22.4 mg/ml
(range 0.1–62.9 mg/ml) for feces. The amounts of both meconium
and feces produced by the newborns vary greatly. The total amount of
amniotic ﬂuid is, however, much larger. With an average total volume
of 700 ml at birth, the amniotic ﬂuid contains ∼1.5 mg of SALSA
(36). Case 1 showed very high levels of SALSA in meconium.
However, in this case there were no abnormalities during the preg-
nancy, delivery, or the ﬁrst week of life.
Analysis of peptide compositions
Based on the proteomics data, we were able to further analyze
the polypeptide compositions of SALSA molecules from different
sources. Fig. 3 shows a heat map of the 28 identiﬁed peptides
matching to SALSA. For each sample, the relative quantities of
the peptides found within the sample are displayed. Based on this,
the samples were clustered. Very interestingly, the AF samples
clustered together (the left side of the heat map), whereas meco-
nium samples clustered into another distinct group (right side).
Fecal samples were spread out across the horizontal axis. How-
ever, except for F1, F4, and F6, most of the fecal samples clus-
tered separately from the AF samples.
Peptides 7–9, 10, and 25 (marked with arrows in Fig. 3; dis-
played in full in Table I) were most signiﬁcantly different in their
relative abundance (p,0.00002) in AF compared with the in-
testinal samples. Thus, they were analyzed in more detail. Pep-
tides 10 and 25 were relatively more abundant in the AF samples,
whereas peptides 7–9 were relatively less abundant in the AF
samples. The speciﬁc clustering indicates a difference in the
SALSA proteins from AF versus those in meconium and feces
in the particular areas of the protein containing these peptide
sequences. Alternatively, the proteins could become differentially
processed in the different environments, for example, by proteo-
lytic or glycolytic enzymes and/or binding partners.
Structural differences of AF versus intestinal SALSA
Comparative structural analyses were carried out to get more in-
sight into the locations of the discriminatory peptides of AF and
intestinal SALSA. In Fig. 4A, the domain organization of SALSA
is displayed (9). The speciﬁc locations of the ﬁve individual
peptides are displayed above the respective domains, where they
are found in the SALSA protein. Peptides 7–9 have the same core
sequence but vary in 1 to 2 aa. There is one representative of
peptide 7, 8, or 9 in every SRCR domain except in domain 14.
FIGURE 1. SALSA in AF, meconium, and infant feces. SDS-PAGE
comparison of SALSA in protein extracts from AF, meconium (M), and
fecal (F) samples from 1-wk-old infants. Examples from three out of nine
different cases are shown. For comparison, SALSA in saliva (S) from
a healthy adult is shown. The samples were subjected to SDS-PAGE under
nonreducing conditions, transferred to a nitrocellulose membrane, and
probed with a monoclonal anti-SALSA Ab. Protein extracts were prepared
from 50 ml AF or 125 mg meconium and feces and diluted 1:10–1:100
before loading. Because of differences in size and glycosylation, the SALSA
proteins appear as bands and smears of variable sizes.
The Journal of Immunology 3
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Thus, the differences in these peptides simply represent variations
in the SRCR domains. Peptide 10 was found within all SRCR
domains, again with the exception of domain 14, whereas peptide 25
was only found once in the entire protein, within the ZP domain.
The three-dimensional structures of some SRCR and ZP
domains have been determined, allowing comparison between
the peptide sequences and the structures. In Fig. 4B, the crystal
structure of the group A SRCR of the Mac2-binding protein is
displayed (37). Peptide 10, present in SRCR domains 1–13 and
found to be more abundant in the AF SALSA, is located in an
extended loop between aa 56 and 69 in the SRCR model do-
main of the Mac2-binding protein. No known function has been
ascribed to this particular loop. However, when the remaining
peptides (7–9, 25) were compared with the homologous SRCR
and ZP structures, they could be potentially linked to estab-
lished SALSA functions. SALSA is known to bind to a broad
range of bacteria, and a very speciﬁc peptide sequence respon-
sible for these interactions, RVEVLYxxxSW, has been identi-
ﬁed within the SRCR domain (21). This sequence is highlighted
in yellow (68% homology to SALSA) in Fig. 4B. Interestingly,
peptides 7–9 (highlighted in red), which were relatively less
abundant in AF, overlapped with the bacterial binding sequence.
The N-terminal QSW of these peptides matched the xSW from
the bacterial binding sequence. Theoverlapisshowninorange
in Fig. 4B.
In Fig. 4C, the crystal structure of a full-length ZP domain from
chicken sperm receptor 3 (cZP3) is displayed (38). This homol-
ogous structure was used as a model for the SALSA ZP domain.
The two proteins are similar (56% identity) in the region of
peptide 25. The sequence of the AF-abundant SALSA peptide
matched a loop extending from the ZP-c subdomain. An analysis
of the cZP3 structure suggested that this area is directly involved
in the ZP domain–mediated protein dimerization function, thus
providing the speciﬁcity of the egg coat assembly (38). Due to
the high level of similarity, we suspect that the same is the case
for SALSA. Our analysis of the abundance of certain peptide
sequences thus suggests the existence of structural differences
between AF- and intestine-derived (meconium and fecal) SALSA
in regions with a direct link to suggested protein functions
(i.e., bacterial binding via the SRCR domain and extracellular
communication/polymerization via the ZP domain).
Functional differences between AF and intestinal SALSA
To directly address the question whether AF and intestinal
SALSA have functional differences, we tested their binding to
three different types of streptococci, GAS, GBS, S. gordonii,
E. coli (an intestinal commensal), and S. Typhimurium (an in-
testinal pathogen). The bacteria were incubated with protein
extracts diluted into a SALSA concentration of 0.5 mg/ml. After
centrifugation, the bound SALSA was eluted using SDS-PAGE
loading buffer containing 10 mM EDTA. The ability of the
bacteria to absorb SALSA from the various solutions is visu-
alized in Fig. 5 by Western blotting. The binding patterns are
representatives of two to six tested individuals.
SALSA from AF bound to GAS, GBS, S. gordonii, and E. coli.
The SALSA band disappeared after incubation with the bacteria
in all cases and was found after elution from the bacterial sur-
faces. In contrast, SALSA from meconium did not bind to any of
the bacterial strains but remained in the solution. Interestingly,
S. Typhimurium did not bind any type of SALSA. The fecal sam-
ples showed greater variation in the bacterial binding abilities.
From some samples, SALSA did not bind to any of the tested
bacteria. However, from other samples, SALSA bound to GAS
and S. gordonii. Some binding was also observed to GBS. How-
ever, binding was not strong enough to deplete SALSA from the
fecal suspension used in the assay. To control that no factors in
meconium were inhibiting the SALSA binding, AF was mixed with
varying amounts of meconium. Regardless of the concentration of
meconium, the same amount of SALSA became bound and was
eluted from the bacteria, showing no effect of meconium products
on the binding of AF-SALSA to the bacteria (data not shown).
Given the clear variation in binding abilities of SALSA from AF
and meconium to bacteria, we further tested SALSA interactions with
known endogenous protein ligands of salivary SALSA. We compared
SALSA binding from several AF and meconium samples to secretory
IgA, MBL, and C1q (Fig. 6). SALSA from all AF samples bound
clearly to IgA, MBL, and C1q. No binding to any of the ligands was
After performing a screen of various body ﬂuids and biological
materials from mucosal surfaces, we were able to identify the
SALSA protein in AF and meconium. To our knowledge, this is the
FIGURE 2. SALSA levels in AF, meconium, and infant feces. The concentrations of SALSA in AF, meconium, and feces samples as determined by LC-
MS/MS (A) or ELISA (B). (A) The total amount of peptides matched to SALSA was compared with the overall amount of identiﬁed peptides. Lower
relative levels of SALSA were seen in AF samples compared with meconium and feces. (B) Actual SALSA concentrations determined by ELISA.
Measurements were performed in duplicates and repeated three times.
4 THE SALIVARY SCAVENGER AND AGGLUTININ (SALSA) IN EARLY LIFE
ﬁrst description of the presence of SALSA in these biological
materials. Previous studies have attributed a role for SALSA in
both innate immunity and epithelial differentiation at mucosal
surfaces (10). Given the high degree of cell differentiation in
general during fetal maturation and the importance of innate im-
munity at both the fetal and neonatal stages, SALSA may play key
roles in these processes during early life. During prenatal life, AF
is constantly surrounding the fetal skin and mucosal surfaces of
the nasal and gastrointestinal tract. To get a broad understanding
of SALSA in early life, we included samples from mucosal sur-
faces at both the fetal stage (AF) and the infant stages (meconium
and fecal samples).
A key ﬁnding of the current study was the abundance of SALSA
in the meconium and feces in early infancy, averaging ∼3to4%
(range 1–10%) of the total protein in these samples. Such abun-
dance underlines the importance of this protein at this early stage
of life. Both the relative abundance studies and the actual con-
centration measurements of SALSA revealed that the levels of
SALSA in the extracts from meconium and fecal samples were
notably higher than those in the AF samples. On the average, the
concentrations of SALSA were 2.1 mg/ml for AF, 45.8 mg/ml for
meconium, and 22.4 mg/ml for feces. However, AF constitutes one
of the largest physiological reservoirs of SALSA (∼1.5 mg) be-
cause of its large volume during late pregnancy (∼700 ml at birth)
(36). After birth, the largest pool of SALSA apparently resides in
the gastrointestinal tract (9, 10).
Initially, Western blotting was used to analyze potential varia-
tions in the size of the SALSA protein among individuals and
among the different types of samples collected. A band was ob-
served in the area of 340 kDa for all AF, meconium, and fecal
samples (Fig. 1). This correlated well to observations made in
other body ﬂuids such as saliva and lacrimal ﬂuid (Fig. 1) (7, 14).
Unfortunately, direct comparison with SALSA in saliva from the
individuals in this cohort was not possible, because salivary
samples were not available for the study. Individual differences
were observed when the same types of biological samples from
different individuals were compared, especially in meconium and
feces. Size polymorphisms of SALSA have been described before
and used to classify individuals into four groups (groups I–IV)
(20, 33). This grouping was shown to correlate with Lewis-Ag
expression, secretor status, and bacterial binding properties (33).
Individual variations in SALSA observed in AF, meconium, or
feces may reﬂect such grouping.
Differences in the SALSA band patterns across different tissues
within the same individual were also observed in this study. Pre-
viously, two isoforms of SALSA have been described in tear ﬂuid
from the same individual, but SALSA has never before been
compared in different mucosal compartments from the same person
(14). In this study, a clear difference among the proteins found
in AF, meconium, and feces were observed. Overall, SALSA in
meconium appeared with a lower m.w. than that in AF. The same
seemed to be the case for fecal SALSA. However, the difference
to AF SALSA was not so clear cut. The origin of SALSA, both in
the fetal gut and within the amniotic cavity, remains unclear. AF is
a bioactive medium with constituents actively secreted by cells
lining the amniotic cavity and, during early stages, liquid ﬁltered
from the maternal blood. As gestation progresses, AF includes
a signiﬁcant volume from fetal urine (up to 50%). However,
SALSA could not be detected in adult urine samples (data not
shown). SALSA in AF may be secreted into AF by either the
amniotic epithelial cells or the prenatal fetus. Meconium is to a
great extent a concentrate of the swallowed AF during the fetal
development, and AF could thus be the source of SALSA in
meconium. In the infant intestine, as well as during later stages,
production by the intestinal epithelium is probably the most im-
portant source of SALSA.
In addition to Western blotting, an analysis using proteomics
techniques revealed peptide variations in functionally relevant
regions of the SALSA protein. A mass spectrometric analysis of the
relative abundances of the 28 SALSA-related peptides revealed
sample-speciﬁc patterns. Peptides of AF samples clustered clearly
separately from the meconium and fecal ones. Peptides 7–10 and
Table I. Peptide sequences matched to SALSA
Peptide No. Sequence
Speciﬁc amino acid sequences of peptides matching to SALSA in the LC-MS/MS
analysis. Displayed are only the peptides with a very signiﬁcantly different rela-
tive abundance in AF and meconium/feces ( p,0.00002). X indicates leucine or
FIGURE 3. Semiquantitative heat map proteomics analysis of peptides
matched to SALSA. Samples were obtained from nine individual infants.
The graph displays the abundance of 28 peptides matching to SALSA,
numbered on the right-hand side. The color coding indicates how abundant
a particular peptide is in one particular sample (red, very abundant; blue,
less abundant). Based on these relative amounts, the samples have been
clustered (top horizontal axis). All AF samples cluster to the left side of
the heat map together with fecal samples F1, F4, and F6. A differential
abundance between the AF group and the intestinal samples is particularly
observed for ﬁve peptides: 7, 8, 9, 10, and 25 (marked with red arrows).
The sequences of the respective peptides are shown in Table I.
The Journal of Immunology 5
25 together cover three distinct regions within the SALSA protein
(Fig. 4). Peptides 7–9 were relatively less abundant in the AF
SALSA compared with intestinal SALSA, whereas peptides 10
and 25 were relatively more abundant. It has long been known that
SALSA binds to and agglutinates several types of bacteria in the
mouth and in the gut (16–21). The broad bacterial binding prop-
erty has been assigned to one particular peptide sequence found
in the SRCR domains (RVEVLYxxxSW) (21). Three amino acids
(xSW) in this sequence overlap with the region that was found to
be relatively more abundant in the intestinal SALSA than in AF
SALSA. Such a structural difference could indicate an altered
bacterial binding ability of the intestinal SALSA and/or a difference
in the structural integrity of the intestinal and AF forms of SALSA.
A difference became even more apparent when the peptide
enriched in the AF samples (peptide 25) was considered. The
sequence of this peptide was found in the ZP domain only. ZP
domains are found in hundreds of extracellular matrix proteins.
Often these proteins polymerize through the ZP domain into ﬁbrils
or matrices and aid in transforming the cell shape and creating
polarization (39). This has been shown for hensin, the SALSA-
homolog in rabbits (40). Dmbt1-knockout mouse embryos were
found unable to induce columnar epithelia, which is a critical step
during embryonic development. As a consequence, the mice died
at an early time point (41, 42). Polymerization of SALSA through
the ZP domain could as well affect the avidity of interactions with
bacteria in the gut. Both soluble and extracellular matrix–bound
SALSA would be monomeric in the absence or by the cleavage of
the ZP domain. This could weaken and even abolish the bacterial
binding ability. The apparent presence or absence of the ZP do-
main in AF SALSA and intestinal SALSA, respectively, could
therefore easily have functional relevance.
Despite the different relative abundances of peptides of the
various forms of SALSA, differences at the primary amino acid
sequence level are unlikely. Peptides 7–10 are all located within
the highly conserved SRCR domains. Each SRCR domain is
expressed from a single exon, and therefore, variations within the
domains are not expected. However, at the transcriptional level,
several isoforms have been described, all showing varying num-
FIGURE 4. Structural overview of the SALSA protein. The peptides highlighted in Fig. 3 and Table I were located in the structure of the SALSA protein.
(A) SALSA is comprised of 13 N-terminal SRCR domains, 2 C1r/C1s Uegf Bmp-1 domains surrounding the 14th SRCR domain, and a C-terminal ZP
domain (9). The peptides with varying abundance are displayed above the domains of SALSA where they were found. The relative abundances of the
peptides found in AF or intestinal (meconium and fecal) samples are indicated. The peptides relatively less abundant in AF samples (peptides 7–9) all
represent a particular sequence of the SRCR domain. This sequence is present in all SRCR domains except 14. Peptides 10 and 25 were most abundant in
the AF SALSA protein. Peptide 10 was found in each SRCR domain except domain 14, and peptide 25 was found only in the ZP domain. (B) Location of
peptides 7–9 (red) in a crystal structure representation of the SRCR domain of Mac-2 binding protein (37). These peptides are partially overlapping with the
SRCR peptide sequence responsible for the bacterial binding of SALSA (yellow). The three peptides 7–9 are identical in their N-terminal region. This
region overlaps with the GSW of the bacterial binding sequence (orange). (C) Location of peptide 25 (purple) in a crystal structure model of cZP3 (38). This
sequence is part of the ZP-c subdomain, a structure believed to be directly involved in the protein dimerization function mediated by the ZP domain.
6 THE SALIVARY SCAVENGER AND AGGLUTININ (SALSA) IN EARLY LIFE
bers of SRCR domains. All mRNA transcripts and proteins ana-
lyzed in various tissues by others, so far, have been found to
display a ZP domain (10, 31).
To study whether the observed structural differences are related
to actual functional differences, we tested the ability of AF SALSA,
meconium SALSA, and fecal SALSA to bind to various bacterial
strains. We found that AF SALSA bound strongly to GAS, GBS,
S. gordonii, and E. coli. In contrast, SALSA in meconium was not
able to bind to any of these bacteria. SALSA proteins from the
various fecal samples differed in their binding ability. Some bound
to GAS and S. gordonii, whereas others did not. Still, in the fecal
samples SALSA did not bind to GBS. S. Typhimurium did not bind
SALSA from any of the samples. S. Typhimurium is a known
intestinal pathogen, and GBS is a pathogen especially for the
newborn (43). Interestingly, these two pathogenic strains bound
only little SALSA or none at all. This warrants comparative studies
in larger panels of pathogens and nonpathogens.
In addition to the differences in bacterial binding, we also ob-
served differences in binding to endogenous ligands by SALSA
from AF and meconium. SALSA from AF bound, as expected, to
IgA, MBL, and C1q. However, SALSA from meconium was not
able to bind to any of the tested proteins. These data show that
the structural differences can affect the function of the SALSA
protein. Apparently, SALSA in AF is intact and fully functional.
However, SALSA recovered from meconium and some fecal
samples is altered and does not have the bacterial and endogenous
protein binding property.
The contribution of amniotic cavity and fetal/infant gut to the
total SALSA concentration in the analyzed samples may vary on an
individual basis. As a consequence, site-speciﬁc polymorphisms,
cleavage patterns, oligomerization, and/or glycosylation and other
posttranslational modiﬁcations may explain the SALSA protein
variations among the different types of samples analyzed in this
study. Glycosylation and other posttranslational modiﬁcations may
interfere with trypsin digestion. These have been described in
SALSA from bronchoalveolar lavage, saliva, and lacrimal ﬂuid (44,
45). The size variations may therefore be related to differential
posttranslational modiﬁcations within the different protein areas
making them more or less susceptible to cleavage by trypsin.
Trypsin is naturally present in the intestine and could thus inﬂu-
ence the bacterial binding properties of SALSA in vivo (46). The
ZP domain could be proteolytically cleaved in the intestinal en-
vironment and thus be lost during the protein extraction.
A prominent role for SALSA at the mucosal surfaces has been
suggested, including an involvement in inﬂammatory conditions
such as Crohn disease and ulcerative colitis (47). For infants, the
importance has also been shown through its altered regulation in
preterm infants with neonatal infections (48). In the current study,
we describe for the ﬁrst time, to our knowledge, the presence of
SALSA in both AF and meconium. The very high protein levels
observed support a crucial role for SALSA in the early life. Innate
immune clearance functions and cell-differentiation processes are
vital for fetal development, and SALSA may be involved in both.
SALSA in AF could be involved in epithelial cell differentiation
and extracellular matrix binding through the ZP domain. At the
same time, it could play an important role in keeping the amniotic
cavity sterile by agglutinating bacteria and activating the com-
plement system. The observed loss of bacterial and endogenous
FIGURE 5. Differences in bacterial binding abilities among SALSA
from AF, meconium, and feces. The ability of SALSA to bind GAS, GBS,
and S. gordonii was compared for SALSA in AF (A), meconium (B), and
feces (Cand D). SALSA binding to the intestinal microbes E. coli and
S. Typhimurium was also compared for SALSA in AF (E) and meconium
(F). Lane 1 shows SALSA in the initial sample. Lanes 2,4, and 6show
SALSA in the supernatants after incubation with GAS, GBS, and
S. gordonii (A–D)orS. Typhimurium and E. coli (Eand F), respectively.
The lack of a SALSA band indicates binding to the bacterial surface. Lanes
3,5, and 7show SALSA eluted from the surfaces of GAS, GBS, and
S. gordonii (A–D). Lanes 3 and 5in (E) and (F) show SALSA eluted from
S. Typhimurium and E. coli, respectively. SALSA from AF binds to GAS,
GBS, S. gordonii, and E. coli, whereas SALSA from meconium does not
bind to any of the tested bacteria. SALSA from feces showed individual
differences in the ability to bind bacteria. (C) shows SALSA binding to
GAS and S. gordonii, but less to GBS. In (D), no binding of SALSA to any
of the tested bacteria is seen. SALSA did not bind S. Typhimurium. For
each sample type, two to six individuals were tested.
FIGURE 6. A difference between SALSA from AF and meconium in
binding to endogenous protein ligands of salivary SALSA. The binding of
SALSA from AF and meconium (M) to IgA, MBL, and C1q was tested by
ELISA. SALSA from every AF sample, but from none of the meconium
samples, bound to IgA, MBL, and C1q. The data shown are averages of
The Journal of Immunology 7
ligand binding in the intestine may indeed be a result of regulation
of the protein function at the mucosal surface (e.g., by proteolytic
or glycolytic cleavage). In the early neonatal life, the mucosal
surfaces throughout the body undergo massive transformation.
The initial colonization of the body starts, and a commensal
healthy microbiome is acquired in concert with the development
of the body’s own immune system. The differences in bacterial
binding of SALSA, observed in the fecal samples, may be part of
the regulation of the colonizing microbiota. The very early pres-
ence of SALSA and its bacterial binding functions suggest that it
may be a key player in the selection of a healthy intestinal com-
We thank the volunteers for participation in this study and Antti Nuikka
for expert technical assistance. Recombinant MBL was a kind gift from
Prof. Steffen Thiel (Aarhus University, Aarhus, Denmark). Assistance
with the structural modeling was kindly provided by Asst. Prof. Sakari
Jokiranta (University of Helsinki, Helsinki, Finland).
The authors have no ﬁnancial conﬂicts of interest.
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