SALSA: A Regulator of the early Steps of Complement Activation on Mucosal Surfaces

Abstract

Complement is present mainly in blood. However, following mechanical damage or inflammation, serous exudates enter the mucosal surfaces. Here the complement proteins interact with other endogenous molecules to keep microbes from entering the parenteral tissues. One of the mucosal proteins known to interact with the early complement components of both the classical and the lectin pathway, is the salivary scavenger and agglutinin (SALSA). SALSA is also known as DMBT1 (deleted in malignant brain tumors 1) and gp340. It is found both attached to the epithelium and secreted into the surrounding fluids of most mucosal surfaces. SALSA has been shown to bind directly to C1q, mannose binding lectin (MBL) and the ficolins. Through these interactions SALSA regulates activation of the complement system. In addition, SALSA interacts with surfactant proteins A and D, secretory IgA and lactoferrin. Ulcerative colitis and Crohn’s disease are examples of diseases, where complement activation in mucosal tissues may occur. This review describes the latest advances in our understanding of how the early complement components interact with the SALSA molecule. Furthermore, we discuss how these interactions may affect disease propagation on mucosal surfaces in immunological and inflammatory diseases.

Full text

March 2016 | Volume 7 | Article 851
MINI REVIEW
published: 08 March 2016
doi: 10.3389/mmu.2016.00085
Frontiers in Immunology | www.frontiersin.org
Edited by:
Michael C. Carroll,
Boston Children’s Hospital and
Harvard Medical School, USA
Reviewed by:
Masahide Tone,
Cedars-Sinai Medical Center, USA
Matthew Cook,
Canberra Hospital and Australian
National University, Australia
*Correspondence:
Seppo Meri
seppo.meri@helsinki.
Specialty section:
This article was submitted to
Immunological Tolerance,
a section of the journal
Frontiers in Immunology
Received: 02November2015
Accepted: 22February2016
Published: 08March2016
Citation:
ReichhardtMP and MeriS (2016)
SALSA: A Regulator of the Early
Steps of Complement Activation on
Mucosal Surfaces.
Front. Immunol. 7:85.
doi: 10.3389/mmu.2016.00085
SALSA: A Regulator of the Early
Steps of Complement Activation on
Mucosal Surfaces
Martin Parnov Reichhardt and Seppo Meri*
Immunobiology Research Program, Research Programs Unit, Department of Bacteriology and Immunology, Haartman
Institute, University of Helsinki, Helsinki, Finland
Complement is present mainly in blood. However, following mechanical damage or
inammation, serous exudates enter the mucosal surfaces. Here, the complement
proteins interact with other endogenous molecules to keep microbes from entering
the parenteral tissues. One of the mucosal proteins known to interact with the early
complement components of both the classical and the lectin pathway is the salivary
scavenger and agglutinin (SALSA). SALSA is also known as deleted in malignant brain
tumors 1 and gp340. It is found both attached to the epithelium and secreted into the
surrounding uids of most mucosal surfaces. SALSA has been shown to bind directly to
C1q, mannose-binding lectin, and the colins. Through these interactions SALSA regu-
lates activation of the complement system. In addition, SALSA interacts with surfactant
proteins A and D, secretory IgA, and lactoferrin. Ulcerative colitis and Crohn’s disease are
examples of diseases, where complement activation in mucosal tissues may occur. This
review describes the latest advances in our understanding of how the early complement
components interact with the SALSA molecule. Furthermore, we discuss how these
interactions may affect disease propagation on mucosal surfaces in immunological and
inammatory diseases.
Keywords: gp340, DMBT1, Crohn’s disease, colins, MBL, C1q, ulcerative colitis, IBD
INTRODUCTION
Activation of the complement system is strongly involved in generating inammation, combatting
microbial infections, and participating in clearance of non-viable tissue. Although complement is
present mainly in blood, it is also found in serous exudates on mucosal surfaces, such as in the oral
cavity or the airways (1, 2). is is particularly seen aer mechanical, infectious, or immune damage,
e.g., in periodontal disease or SLE (3). When serous exudates enter the mucosal surfaces, innate
immune proteins interact with mucosal surface proteins. Together, these molecules participate in
clearance and defense against invading microorganisms. Although bleeding at the mucosal surfaces
is observed daily, even in healthy individuals, the role of the complement system in this environment
has so far been studied very little. Of particular interest would be the need to suppress complement-
mediated inammation, while still mediating the antimicrobial defense functions.

TABLE 1 | Endogenous and microbial ligands of SALSA.
Endogenous
ligand
Suggested functional relevance
C1q Complement regulation (4)
MBL Complement regulation (6)
Ficolins Complement regulation (6)
SpD Microbial agglutination (9)
SpA Microbial agglutination (16)
IgA Microbial agglutination (8)
Lactoferrin Bacterial binding (20)
DNA Inammation (21)
Heparan sulfate Inammation (21)
Trefoil factors Tissue homeostasis (17)
MUC5B Microbial agglutination (22)
Fibrin Not known (19)
Fibrinogen Not known (19)
Erythrocytes Aggregation (19)
Platelets Aggregation (19)
Microbe Specic strains
Streptococcus S. pyogenes, S. agalactiae, S. pneumonia, S. mutans, S. mitis,
S. oralis, S. salivarius, S. gordonii, S. crista, S. parasanguinis,
S. vestibularis, S. intermedius, S. anginosus, S. suis (7, 23–25)
Lactobacillus L. rhamnosus, L. casei, L. reuteri, L. lactis (26)
Other bacteria Staphylococcus aureus, Bidobacterium, Actinomyces,
Salmonella enterica serovar Typhimurium, Helicobacter pylori,
Haemophilus inuenzae, Klebsiella oxytoca (23–28)
Viruses HIV, IAV (29, 30)
The listed ligands have been found to bind either human SALSA, the murine-ortholog
of SALSA, or the recombinantly expressed bacterial-binding peptide, SRCRP2.
March 2016 | Volume 7 | Article 852
Reichhardt and Meri
SALSA and Complement Activation
Frontiers in Immunology | www.frontiersin.org
Salivary Scavenger and Agglutinin
One of the molecules at the mucosal surfaces that interact with the
early complement components is a protein that we named salivary
scavenger and agglutinin (SALSA) (4–7). SALSA, also known as
gp340, “deleted in malignant brain tumors 1” (DMBT1), and sali-
vary agglutinin (SAG), was rst described as a 300–400kDa strep-
tococcus agglutinating agent from saliva (8–10). Subsequently,
SALSA has been suggested to function in epithelial homeostasis,
innate immunity, inammation, and tumor suppression (11–13).
Many of these functions are mediated through interactions with
endogenous ligands. SALSA has been shown to bind the comple-
ment components C1q, mannose-binding lectin (MBL), and the
colins (4, 6). Furthermore, SALSA has been found to interact
with surfactant proteins A and D (SpA and SpD, respectively),
secretory IgA, lactoferrin, brin/brinogen, trefoil factors, and
mucin-5B (Tab le 1 ) (9, 14–19). e multiple binding partners
suggest that SALSA plays a central role in regulating inamma-
tion and immune responses on mucosal surfaces.
SALSA in Antimicrobial Defense
Salivary scavenger and agglutinin is expressed at most mucosal
surfaces, including the lungs, oral cavity, gastrointestinal tract,
and vagina (31–35). It has been found both attached to the epi-
thelium and secreted into the lining uids, such as saliva, tear
uid, and respiratory mucosal secretions (8, 9, 14, 36). Recent
studies detected SALSA in the amniotic uid and in the intestines
of neonates (37). SALSA was estimated to constitute up to 10% of
the total protein amount in meconium and in the saliva of young
children (<3years), making it one of the most abundant proteins
in these environments (37, 38).
On the mucosal surfaces, SALSA has been shown to regulate
the local immune system. On one hand, it scavenges invading
microorganisms, whereas, on the other hand, it interacts with the
mucosal epithelium to improve the integrity of this physical bar-
rier (Figure1A) (13, 39). SALSA binds a broad range of microbes,
including viruses and bacteria (Table1). Studies have shown that
SALSA in the oral and intestinal mucosal secretions is sucient
to suppress infection by agglutinating microorganisms and keep-
ing them from infecting the tissue. is has been observed for
Salmonella enterica, HIV-1, and inuenza A-virus (IAV) (27, 29,
30, 40). ese studies suggested that SALSA simply functioned by
agglutinating the microbes. However, the role of SALSA appears
to be more complex than that. SALSA binds, e.g., to epithelial and
tooth surfaces in addition to being secreted into the uid phase
(23). e epithelium-attached localization of a protein with a solely
bacteria-agglutinating function would not appear to be benecial
for the human host. is paradox has been made clear by studies
showing that SALSA, in some cases, may actually be exploited
by the invading microbes. A study of dental caries showed that
certain variants of the SALSA protein correlated positively with
Streptococcus mutans adhesion to SALSA-coated hydroxyapatite
surfaces and the development of dental caries. Other SALSA
variants displayed the opposite correlation (41). In the case of
HIV-1 infection, the salivary uid SALSA protein was found to
interfere with oral transmission. However, SALSA expressed on
the vaginal epithelium had an enhancing eect on the infectivity
of the virus (35). ese ndings suggest that some microbes have
evolved mechanisms to utilize SALSA to infect the human body.
Isoforms of the SALSA Protein
As indicated above, various variants of SALSA may interact dier-
ently with microbes. Indeed, dierent SALSA isoforms have been
identied on various mucosal surfaces. ese have been shown to
vary both in protein sequence and in the glycosylation patterns
(23, 36, 37). e gene for SALSA (in chromosome 10q26.13)
encodes 13 highly conserved scavenger receptor cysteine-rich
(SRCR) domains. ese 109-amino acid-long motifs are found
as “pearls on a string” separated by SRCR interspersed domains
(SIDs) (Figure1B). e stretch of 13 SRCR domains is followed
by 2 C1r/C1s, urchin embryonic growth factor and bone morpho-
genetic protein-1 (CUB) domains encompassing the 14th SRCR
domain. Finally, a zona pellucida (ZP) domain is found at the
most C-terminal end (31, 42). e repetitive sequence of SRCR
domains may facilitate alternative splicing (43). Indeed, mRNA
transcripts encoding between 8 and 13 of the N-terminal SRCR
domains have been observed, all in all revealing up to 7 distinct
alleles (31, 42, 44). It has been estimated that SALSA contains
25–45% (w/w) of carbohydrate (8, 31). SALSA contains all the
major sugar components. However, dierences have been found
to correlate to the secretor [Se(+/−)] status (±expression of the
α1-2fucosyl-transferase). e blood group antigens, ABO, and
Lewis antigens b and y (Leb and Ley) were found on SALSA from
Se(+) individuals. In contrast, SALSA from Se(−) individuals did
not contain ABO, Leb nor Ley antigens. Instead, Lewis antigens a

SRCR domain
CUB domain ZP domain
SRCR interspersed domain
A
B
IgA and IgG
C1q/MBL/ficolins
SALSA
Epithelial damage
Healthy epithelium
SpA/SpD
Lume
n
Tissue
Microbe
IIIIII IV
Epithelial cell
FIGURE 1 | Function and structure of SALSA at the mucosal surfaces. (A) At the mucosal surfaces, the SALSA protein is mainly found associated with the
epithelium and secreted into the surrounding uids. The known features and functions of SALSA are presented in four panels (I–IV). (I) SALSA is present on the
epithelial cell surface and deposited in the extracellular matrix, where it is involved in maintaining epithelial homeostasis. (II) Fluid-phase SALSA binds a broad array
of microbes. It has been shown to agglutinate viruses, as well as both Gram-positive and Gram-negative bacteria thus preventing them from invading the parenteral
spaces. (III) SALSA interacts with other endogenous molecules present at the mucosal surfaces, such as surfactant proteins SpA and SpD as well as IgA. It is
believed that these molecules cooperate in antimicrobial defense. (IV) In the case of epithelial damage, cells and molecules from the tissue become mixed with the
luminal contents. In this context, SALSA may bind the complement sensor molecules C1q, MBL, and the colins, thereby SALSA could initiate complement
activation against distinct microbes or participate in the clearance of injured tissue components. (B) In its molecular structure, SALSA contains a stretch of 13
scavenger receptor cysteine-rich (SRCR) domains separated by SRCR interspersed domains. These are followed by two C1r/C1s, urchin embryonic growth factor
and bone morphogenetic protein-1 (CUB) domains surrounding the 14th SRCR domain. Finally, a zona pellucida (ZP) domain is found at the most C-terminal end of
the protein.
March 2016 | Volume 7 | Article 853
Reichhardt and Meri
SALSA and Complement Activation
Frontiers in Immunology | www.frontiersin.org
and x (Lea and Lex) were present (45, 46). us, dierent forms
of the SALSA protein exist. ey are produced both by variations
in the protein chain and in the extent and nature of glycosyla-
tion. e SALSA protein composition varies not only between
individuals but also in dierent body compartments within the
same individual (23, 36, 37).
SALSA AND COMPLEMENT
Interactions of C1q, MBL, and Ficolins
with SALSA
C1q, MBL, and colins all form bouquet-like structures, where
each subunit contains a collagen-like domain (stalk) and a carboxy-
terminal globular domain (the “ower”) (47, 48). C1q binds speci-
cally to surface-attached IgG and IgM. However, other endogenous
non-immunoglobulin ligands have been found, including SALSA
(4, 6). Also, MBL, M-colin, H-colin, and L-colin were found
to bind to SALSA (6). All interactions between SALSA and the
complement molecules were calcium dependent.
C1q was shown to bind SALSA through the globular domain in
a region close to the immunoglobulin-binding site (49). Similarly,
it appears that MBL utilizes the globular carbohydrate recognition
domain (CRD) for the interaction with SALSA. Due to the heavy
glycosylation of SALSA, sugar structures may function as a target
for the CRD of MBL (8, 31). When the binding of MBL was tested
to SALSA puried from the saliva of a single donor up to 60%
inhibition of the SALSA–MBL interaction was observed when
5 mM fucose was added to the uid phase (7). MBL binds to
the Leb antigen, a fucose-containing oligosaccharide (7). A clear
dierence was observed in the binding of MBL to SALSA from
secretors vs. non-secretors (7). is correlates to the nding that
only SALSA from Se(+) individuals contains the Leb antigen (45,
46). is strongly suggests that MBL binds via the CRD to the Leb
antigen of SALSA.
Complement Activation and Regulation by
SALSA
It has been shown that the binding of C1q to SALSA is sucient to
initiate activation of the classical complement activation pathway
(4, 6). In addition, SALSA was shown to inuence the activation
of the lectin pathway through interactions with MBL and the co-
lins (5, 6). e overall outcome of SALSA-mediated complement
regulation varies with the specic location of SALSA (6). SALSA
coated onto a microtiter plate surface activated complement as

March 2016 | Volume 7 | Article 854
Reichhardt and Meri
SALSA and Complement Activation
Frontiers in Immunology | www.frontiersin.org
measured by deposition of C4b and C3b aer incubation with
normal human serum (NHS). Using MBL-decient serum,
approximately 30% of the total complement activation was lost
(6). e residual activation is likely mediated by C1q and possibly
also by the colins (4, 6). In contrast to complement activation
observed by surface-bound SALSA, SALSA in the uid phase
caused a dose-dependent inhibition of the lectin pathway (6). No
such eect was observed on the classical pathway, which may be
due to the almost 100 times higher concentrations of C1q vs. MBL.
SALSA was able to interfere with the binding of the MBL–MASP2
complex to surface-coated mannan. Candida albicans is a known
target for MBL-mediated complement activation. When NHS was
mixed with increasing concentrations of SALSA and incubated
with C. albicans a dose-dependent inhibition of the deposition of
both C4b and C3b was observed on the Candida surface (6). e
dual eects of SALSA on the complement system appear con-
tradictory at rst glance. On one hand, by binding to MBL and
colins in the uid phase, SALSA can prevent their binding to
targets. On the other hand, when bound to a surface, SALSA can
direct complement activation against appropriate targets, such as
microbes. Overall, it appears that SALSA is a mucosal rst line
recognition molecule that can distinguish between targets to be
cleared vs. structures to be tolerated.
Increased SALSA expression may alone and in concert with,
e.g., C1q and MBL, lead to increased microbial clearance. In addi-
tion to the interactions with the complement proteins, SALSA
can also mediate its anti-bacterial and inammation regulating
functions through interactions with IgA, SpA, and SpD (8, 9, 16).
e functional outcome of these interactions is a cooperative
eect on the microbial agglutination (Figure1A) (50, 51). SALSA,
SpA, and SpD have a dual eect against IAV: viral agglutination
and inammatory modulation (52). e binding of the SALSA
ligand SpD to IAV has been shown to induce a strong respiratory
burst response in neutrophils invitro. is response was reduced
by the addition of SALSA (51). It has been suggested that this
allows a regulated response by the neutrophils, with an increased
uptake of IAV but without an excessive and potentially harmful
burst response (13). A similar feature is observed in the case of
C-reactive protein and the other pentraxins. ey target C1q to
apoptotic and necrotic tissue, while simultaneously recruiting
factor H to limit the complement activation (53, 54). is process
is relevant during the removal of apoptotic debris at the mucosal
surfaces, as well (55). e dierential outcome of the interaction
of SALSA with complement may represent a similar balanced
eector mechanism against invading microbes.
SALSA AND COMPLEMENT IN
INFLAMMATORY BOWEL DISEASE
Intestines are one of the primary sites, where an imbalance
between activation and control of immune responses leads to
disease. Inammatory bowel disease (IBD) encompasses two
chronic relapsing and remitting inammatory conditions of the
gastrointestinal tract. ese are known as ulcerative colitis (UC)
and Crohn’s disease (CD). Together, they aect up to 1:250 in
the adult population (56). In children, the incidence of IBD is on
the rise. Disease onset is during childhood or adolescence for up
to 25% of the patients; although the mortality of the disease has
been declining, it has a major impact on the development of these
young individuals (57). e fundamental causes of the diseases
are still obscure.
Several associations have been found between complement
components and IBD. Specically for the gut mucosa, the devel-
opment of CD has been associated with an altered expression of
components of the lectin pathway. e frequency of the MBL2 gene
allele, which results in MBL deciency, was signicantly elevated
in pediatric patients with CD compared to healthy controls or
adults with Sjögren’s syndrome (58, 59). Deciencies in classical
and alternative pathway components are rarer. Some patients
decient in C1 inhibitor, which is commonly associated with
hereditary angioedema, were found to develop non-infectious
enteritits and IBD (60–62). To further highlight an involvement
of the classical and lectin pathways of complement, we recently
observed an association of pediatric IBD to an MHC haplotype
that involves a deciency of two C4 genes (HLA-A03; HLA-B07;
one C4A gene; one C4B gene; HLA-DRB115) (63).
A study of lectin pathway components during CD treatment
found a dramatic impact on M-colin and MASP-3 levels in
patients responding to anti-TNF-α therapy (64). However, how
the complement components specically aect the local inam-
matory environment of the gut is not clear yet. e above described
interactions with the SALSA molecule present a potential way
for complement to aect a balanced mucosal immunological
response. Current models of CD pathogenesis include an altered
response to the local microbiota, and an increased SALSA expres-
sion has been linked to several of these responses (65). Studies
have shown that SALSA can be strongly induced by various
immunological stimuli (66, 67). e increased levels of SALSA in
the intestinal epithelium of patients with IBD and in the ethmoid
sinusoidal mucosa of patients with chronic sinusitis suggest that
SALSA expression is part of the mucosal inammatory response
(66–68). Furthermore, a study of preterm infants revealed that an
increase in the pulmonary SALSA levels was part of the mucosal
response to neonatal infection (69).
Salivary scavenger and agglutinin expression by the intesti-
nal epithelium is induced by NOD2 and TLR4 activation (27).
However, the outcome of an induced SALSA expression during
IBD may not necessarily lead to enhanced clearance only. Rather,
the interaction of SALSA with several endogenous molecules may
be part of an ecient but limited immunological response. Failure
in these processes could propagate an unbalanced and overactive
local immune response in IBD. It has been shown that the previ-
ously described SALSA isoforms inuence both the interaction
with microbes and the endogenous ligands, such as IgA, C1q,
and MBL (37). Interestingly, the specic bacterial-binding ability
of SALSA has been found to depend not only on the isoform of
the protein but also on the location of the protein. Fluid-phase
SALSA can bind and aggregate some streptococcal strains, while
SALSA coated to a hydroxyapatite surface does not (23). us,
the association with the mucosal epithelium or the secretion
into the lining uids may further aect the local immunological
environment dierently. Finally, the described interaction of
SALSA and trefoil factors, being important in maintaining the

March 2016 | Volume 7 | Article 855
Reichhardt and Meri
SALSA and Complement Activation
Frontiers in Immunology | www.frontiersin.org
mucosal epithelial barrier, has also been suggested to play a role
in IBD (70, 71).
Salivary scavenger and agglutinin may be part of the normal
immunological response of the mucosal epithelium during infec-
tion. Individual variations in the expression of SALSA isoforms
alternate the ability of SALSA to interact with endogenous
ligands, to invade microbes, and perhaps even to induce a limited
burst response in neutrophils. It is therefore not surprising that a
specic SALSA isoform, lacking the ve most N-terminal SRCR
domains, has been associated with CD (65, 67).
We speculate that the individual variations in the SALSA inter-
actions are key in understanding how this molecule could play
a role in shiing the immunological balance toward increased
inammation at the mucosal surfaces, with detrimental eects
for IBD patients.
FUTURE PERSPECTIVES
At the mucosal surfaces, a very tight immunological response
to infection and inammation is essential. e SALSA molecule
is central player interacting with a multitude of endogenous
molecules, invading microbes and the epithelial barrier. Due to
the tightly linked interactions, a balanced function of the SALSA
molecule is key in avoiding an overactive immune response.
e interplay between the various SALSA isoforms, colins,
MBL, and C1q with modied tissue components, carbohydrates,
acetylated molecules, and microbes on mucosal surfaces provides
an interesting area for future research that may open a new under-
standing of mechanisms underlying the development of mucosal
immunological disorders.
AUTHOR CONTRIBUTIONS
Both authors contributed to the design and writing of this
review.
FUNDING
is work was supported by the Helsinki Biomedical Graduate
Program, the Jenni and Antti Wihuri foundation, Helsinki
University Central Hospital funds (EVO), the Sigrid Jusélius
Foundation, Signe and Ane Gyllenberg Foundation, Magnus
Ehrnrooth Foundation, Helsinki University Funds, the
Stockmann Foundation, and the Academy of Finland.
REFERENCES
1. Boackle RJ. e interaction of salivary secretions with the human complement
system–a model for the study of host defense systems on inamed mucosal
surfaces. Crit Rev Oral Biol Med (1991) 2:355–67.
2. Persson CG, Erjefalt I, Alkner U, Baumgarten C, Grei L, Gustafsson B, etal.
Plasma exudation as a rst line respiratory mucosal defence. Clin Exp Allergy
(1991) 21:17–24. doi:10.1111/j.1365-2222.1991.tb00799.x
3. Courts FJ, Boackle RJ, Fudenberg HH, Silverman MS. Detection of functional
complement components in gingival crevicular uid from humans with
periodontal diseases. J Dent Res (1977) 56:327–31. doi:10.1177/0022034577
0560032001
4. Boackle RJ, Connor MH, Vesely J. High molecular weight non-immunoglobu-
lin salivary agglutinins (NIA) bind C1Q globular heads and have the potential
to activate the rst complement component. Mol Immunol (1993) 30:309–19.
doi:10.1016/0161-5890(93)90059-K
5. Leito JT, Ligtenberg AJ, van Houdt M, van den Berg TK, Wouters D. e
bacteria binding glycoprotein salivary agglutinin (SAG/gp340) activates com-
plement via the lectin pathway. Mol Immunol (2011) 49:185–90. doi:10.1016/j.
molimm.2011.08.010
6. Reichhardt MP, Loimaranta V, iel S, Finne J, Meri S, Jarva H. e salivary
scavenger and agglutinin binds MBL and regulates the lectin pathway of com-
plement in solution and on surfaces. Front Immunol (2012) 3:205. doi:10.3389/
mmu.2012.00205
7. Gunput ST, Ligtenberg AJ, Terlouw B, Brouwer M, Veerman EC, Wouters D.
Complement activation by salivary agglutinin is secretor status dependent.
Biol Chem (2015) 396:35–43. doi:10.1515/hsz-2014-0200
8. Ericson T, Rundegren J. Characterization of a salivary agglutinin reacting with
a serotype c strain of Streptococcus mutans. Eur J Biochem (1983) 133:255–61.
doi:10.1111/j.1432-1033.1983.tb07456.x
9. Holmskov U, Lawson P, Teisner B, Tornoe I, Willis AC, Morgan C, et al.
Isolation and characterization of a new member of the scavenger receptor
superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D bind-
ing molecule. J Biol Chem (1997) 272:13743–9. doi:10.1074/jbc.272.21.13743
10. Mollenhauer J, Wiemann S, Scheurlen W, Korn B, Hayashi Y, Wilgenbus
KK, etal. DMBT1, a new member of the SRCR superfamily, on chromosome
10q25.3-26.1 is deleted in malignant brain tumours. Nat Genet (1997) 17:32–9.
doi:10.1038/ng0997-32
11. Kang W, Reid KB. DMBT1, a regulator of mucosal homeostasis through the
linking of mucosal defense and regeneration? FEBS Lett (2003) 540:21–5.
doi:10.1016/S0014-5793(03)00217-5
12. Ligtenberg AJ, Veerman EC, Nieuw Amerongen AV, Mollenhauer J. Salivary
agglutinin/glycoprotein-340/DMBT1: a single molecule with variable compo-
sition and with dierent functions in infection, inammation and cancer. Biol
Chem (2007) 388:1275–89. doi:10.1515/BC.2007.158
13. Madsen J, Mollenhauer J, Holmskov U. Review: gp-340/DMBT1
in mucosal innate immunity. Innate Immun (2010) 16:160–7.
doi:10.1177/1753425910368447
14. ornton DJ, Davies JR, Kirkham S, Gautrey A, Khan N, Richardson PS, etal.
Identication of a nonmucin glycoprotein (gp-340) from a puried respiratory
mucin preparation: evidence for an association involving the MUC5B mucin.
Glycobiology (2001) 11:969–77. doi:10.1093/glycob/11.11.969
15. Rundegren J, Arnold RR. Dierentiation and interaction of secretory immu-
noglobulin A and a calcium-dependent parotid agglutinin for several bacterial
strains. Infect Immun (1987) 55:288–92.
16. Tino MJ, Wright JR. Glycoprotein-340 binds surfactant protein-A (SP-A) and
stimulates alveolar macrophage migration in an SP-A-independent manner.
Am J Respir Cell Mol Biol (1999) 20:759–68. doi:10.1165/ajrcmb.20.4.3439
17. im L, Mortz E. Isolation and characterization of putative trefoil peptide
receptors. Regul Pept (2000) 90:61–8. doi:10.1016/S0167-0115(00)00110-5
18. Oho T, Bikker FJ, Nieuw Amerongen AV, Groenink J. A peptide domain of
bovine milk lactoferrin inhibits the interaction between streptococcal surface
protein antigen and a salivary agglutinin peptide domain. Infect Im mun (2004)
72:6181–4. doi:10.1128/IAI.72.10.6181-6184.2004
19. Muller H, Renner M, Helmke BM, End C, Weiss C, Poeschl J, etal. Deleted in
malignant brain tumors 1 is up-regulated in bacterial endocarditis and binds
to components of vegetations. J orac Cardiovasc Surg (2009) 138:725–32.
doi:10.1016/j.jtcvs.2009.05.021
20. Mitoma M, Oho T, Shimazaki Y, Koga T. Inhibitory eect of bovine milk lactofer-
rin on the interaction between a streptococcal surface protein antigen and human
salivary agglutinin. J Biol Chem (2001) 276:18060–5. doi:10.1074/jbc.M101459200
21. End C, Bikker F, Renner M, Bergmann G, Lyer S, Blaich S, etal. DMBT1 func-
tions as pattern-recognition molecule for poly-sulfated and poly-phosphor-
ylated ligands. Eur J Immunol (2009) 39:833–42. doi:10.1002/eji.200838689
22. Wickstrom C, Christersson C, Davies JR, Carlstedt I. Macromolecular
organization of saliva: identication of ‘insoluble’ MUC5B assemblies and

March 2016 | Volume 7 | Article 856
Reichhardt and Meri
SALSA and Complement Activation
Frontiers in Immunology | www.frontiersin.org
non-mucin proteins in the gel phase. Biochem J (2000) 351(Pt 2):421–8.
doi:10.1042/bj3510421
23. Loimaranta V, Jakubovics NS, Hytonen J, Finne J, Jenkinson HF, Stromberg
N. Fluid- or surface-phase human salivary scavenger protein gp340 exposes
dierent bacterial recognition properties. Infect Immun (2005) 73:2245–52.
doi:10.1128/IAI.73.4.2245-2252.2005
24. Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I, etal.
Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori,
is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem (2000)
275:39860–6. doi:10.1074/jbc.M006928200
25. Madsen J, Tornoe I, Nielsen O, Lausen M, Krebs I, Mollenhauer J, etal.
CRP-ductin, the mouse homologue of gp-340/deleted in malignant brain
tumors 1 (DMBT1), binds Gram-positive and Gram-negative bacteria and
interacts with lung surfactant protein D. Eur J Immunol (2003) 33:2327–36.
doi:10.1002/eji.200323972
26. Haukioja A, Loimaranta V, Tenovuo J. Probiotic bacteria aect the compo-
sition of salivary pellicle and streptococcal adhesion invitro. Oral Microbiol
Immunol (2008) 23:336–43. doi:10.1111/j.1399-302X.2008.00435.x
27. Rosenstiel P, Sina C, End C, Renner M, Lyer S, Till A, etal. Regulation of DMBT1
via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition
and invasion. J Immunol (2007) 178:8203–11. doi:10.4049/jimmunol.178.12.8203
28. Jumblatt MM, Imbert Y, Young WW Jr, Foulks GN, Steele PS, Demuth DR.
Glycoprotein 340 in normal human ocular surface tissues and tear lm. Infect
Immun (2006) 74:4058–63. doi:10.1128/IAI.01951-05
29. Nagashunmugam T, Malamud D, Davis C, Abrams WR, Friedman HM.
Human submandibular saliva inhibits human immunodeciency virus type
1 infection by displacing envelope glycoprotein gp120 from the virus. J Infect
Dis (1998) 178:1635–41. doi:10.1086/314511
30. Hartshorn KL, White MR, Mogues T, Ligtenberg T, Crouch E, Holmskov U.
Lung and salivary scavenger receptor glycoprotein-340 contribute to the host
defense against inuenza A viruses. Am J Physiol Lung Cell Mol Physiol (2003)
285:L1066–76. doi:10.1152/ajplung.00057.2003
31. Holmskov U, Mollenhauer J, Madsen J, Vitved L, Gronlund J, Tornoe I, etal.
Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D.
Proc Natl Acad Sci U S A (1999) 96:10794–9. doi:10.1073/pnas.96.19.10794
32. Mollenhauer J, Herbertz S, Holmskov U, Tolnay M, Krebs I, Merlo A, etal.
DMBT1 encodes a protein involved in the immune defense and in epithelial
dierentiation and is highly unstable in cancer. Cancer Res (2000) 60:1704–10.
33. Mollenhauer J, Herbertz S, Helmke B, Kollender G, Krebs I, Madsen J, etal.
Deleted in malignant brain tumors 1 is a versatile mucin-like molecule likely to
play a dierential role in digestive tract cancer. Cancer Res (2001) 61:8880–6.
34. Kang W, Nielsen O, Fenger C, Madsen J, Hansen S, Tornoe I, et al. e
scavenger receptor, cysteine-rich domain-containing molecule gp-340 is dif-
ferentially regulated in epithelial cell lines by phorbol ester. Clin Exp Immunol
(2002) 130:449–58. doi:10.1046/j.1365-2249.2002.01992.x
35. Stoddard E, Cannon G, Ni H, Kariko K, Capodici J, Malamud D, etal. gp340
expressed on human genital epithelia binds HIV-1 envelope protein and
facilitates viral transmission. J Immunol (2007) 179:3126–32. doi:10.4049/
jimmunol.179.5.3126
36. Schulz BL, Oxley D, Packer NH, Karlsson NG. Identication of two highly
sialylated human tear-uid DMBT1 isoforms: the major high-molecular-mass
glycoproteins in human tears. Biochem J (2002) 366:511–20. doi:10.1042/
bj20011876
37. Reichhardt MP, Jarva H, de Been M, Rodriguez JM, Jimenez Quintana
E, Loimaranta V, et al. e salivary scavenger and agglutinin in early life:
diverse roles in amniotic uid and in the infant intestine. J Immunol (2014)
193:5240–8. doi:10.4049/jimmunol.1401631
38. Sonesson M, Ericson D, Kinnby B, Wickstrom C. Glycoprotein 340 and sialic
acid in minor-gland and whole saliva of children, adolescents, and adults. Eur
J Oral Sci (2011) 119:435–40. doi:10.1111/j.1600-0722.2011.00879.x
39. Ligtenberg AJ, Karlsson NG, Veerman EC. Deleted in malignant brain
tumors-1 protein (DMBT1): a pattern recognition receptor with multiple
binding sites. Int J Mol Sci (2010) 11:5212–33. doi:10.3390/ijms1112521
40. Wu Z, Van Ryk D, Davis C, Abrams WR, Chaiken I, Magnani J, et al.
Salivary agglutinin inhibits HIV type 1 infectivity through interaction
with viral glycoprotein 120. AIDS Res Hum Retroviruses (2003) 19:201–9.
doi:10.1089/088922203763315704
41. Jonasson A, Eriksson C, Jenkinson HF, Kallestal C, Johansson I, Stromberg N.
Innate immunity glycoprotein gp-340 variants may modulate human suscepti-
bility to dental caries. BMC Infect Dis (2007) 7:57. doi:10.1186/1471-2334-7-57
42. Mollenhauer J, Holmskov U, Wiemann S, Krebs I, Herbertz S, Madsen J, etal.
e genomic structure of the DMBT1 gene: evidence for a region with sus-
ceptibility to genomic instability. Oncogene (1999) 18:6233–40. doi:10.1038/
sj.onc.1203071
43. Mollenhauer J, Muller H, Kollender G, Lyer S, Diedrichs L, Helmke B, etal.
e SRCR/SID region of DMBT1 denes a complex multi-allele system
representing the major basis for its variability in cancer. Genes Chromosomes
Cancer (2002) 35:242–55. doi:10.1002/gcc.10115
44. Mollenhauer J, Helmke B, Muller H, Kollender G, Lyer S, Diedrichs L, etal.
Sequential changes of the DMBT1 expression and location in normal lung
tissue and lung carcinomas. Genes Chromosomes Cancer (2002) 35:164–9.
doi:10.1002/gcc.10096
45. Ligtenberg AJ, Veerman EC, Nieuw Amerongen AV. A role for Lewis a anti-
gens on salivary agglutinin in binding to Streptococcus mutans. Antonie Van
Leeuwenhoek (2000) 77:21–30. doi:10.1023/A:1002054810170
46. Eriksson C, Frangsmyr L, Danielsson Niemi L, Loimaranta V, Holmskov
U, Bergman T, et al. Variant size- and glycoforms of the scavenger receptor
cysteine-rich protein gp-340 with dierential bacterial aggregation. Glycoconj
J (2007) 24:131–42. doi:10.1007/s10719-006-9020-1
47. Reid KB, Porter RR. Subunit composition and structure of subcomponent C1q
of the rst component of human complement. Biochem J (1976) 155:19–23.
doi:10.1042/bj1550019
48. Sellar GC, Blake DJ, Reid KB. Characterization and organization of the genes
encoding the A-, B- and C-chains of human complement subcomponent C1q.
e complete derived amino acid sequence of human C1q. Biochem J (1991)
274(Pt 2):481–90. doi:10.1042/bj2740481
49. Kojouharova MS, Tsacheva IG, Tchorbadjieva MI, Reid KB, Kishore U.
Localization of ligand-binding sites on human C1q globular head region using
recombinant globular head fragments and single-chain antibodies. Biochim
Biophys Acta (2003) 1652:64–74. doi:10.1016/j.bbapap.2003.08.003
50. Ligtenberg AJ, Bikker FJ, De Blieck-Hogervorst JM, Veerman EC, Nieuw
Amerongen AV. Binding of salivary agglutinin to IgA. Biochem J (2004)
383:159–64. doi:10.1042/BJ20040265
51. White MR, Crouch E, Vesona J, Tacken PJ, Batenburg JJ, Leth-Larsen R, etal.
Respiratory innate immune proteins dierentially modulate the neutrophil
respiratory burst response to inuenza A virus. Am J Physiol Lung Cell Mol
Physiol (2005) 289:L606–16. doi:10.1152/ajplung.00130.2005
52. White MR, Crouch E, van Eijk M, Hartshorn M, Pemberton L, Tornoe I, etal.
Cooperative anti-inuenza activities of respiratory innate immune proteins
and neuraminidase inhibitor. Am J Physiol Lung Cell Mol Physiol (2005)
288:L831–40. doi:10.1152/ajplung.00365.2004
53. Jarva H, Jokiranta TS, Hellwage J, Zipfel PF, Meri S. Regulation of complement
activation by C-reactive protein: targeting the complement inhibitory activity
of factor H by an interaction with short consensus repeat domains 7 and 8-11.
J Immunol (1999) 163:3957–62.
54. Deban L, Jarva H, Lehtinen MJ, Bottazzi B, Bastone A, Doni A, etal. Binding
of the long pentraxin PTX3 to factor H: interacting domains and function
in the regulation of complement activation. J Immunol (2008) 181:8433–40.
doi:10.4049/jimmunol.181.12.8433
55. Fox S, Ryan KA, Berger AH, Petro K, Das S, Crowe SE, etal. e role of C1q
in recognition of apoptotic epithelial cells and inammatory cytokine pro-
duction by phagocytes during Helicobacter pylori infection. J Inamm (Lond)
(2015) 12:51. doi:10.1186/s12950-015-0098-8
56. Stone MA, Mayberry JF, Baker R. Prevalence and management of inammatory
bowel disease: a cross-sectional study from central England. Eur J Gastroenterol
Hepatol (2003) 15:1275–80. doi:10.1097/00042737-200312000-00004
57. Van Limbergen J, Russell RK, Drummond HE, Aldhous MC, Round NK,
Nimmo ER, etal. Denition of phenotypic characteristics of childhood-on-
set inammatory bowel disease. Gastroenterology (2008) 135:1114–22.
doi:10.1053/j.gastro.2008.06.081
58. Bak-Romaniszyn L, Szala A, Sokolowska A, Mierzwa G, Czerwionka-Szaarska
M, Swierzko AS, etal. Mannan-binding lectin deciency in pediatric patients
with inammatory bowel disease. Scand J Gastroenterol (2011) 46:1275–8. do
i:10.3109/00365521.2011.594087

March 2016 | Volume 7 | Article 857
Reichhardt and Meri
SALSA and Complement Activation
Frontiers in Immunology | www.frontiersin.org
59. Aittoniemi J, Pertovaara M, Hulkkonen J, Pasternack A, Hurme M, Laippala
P, et al. e signicance of mannan-binding lectin gene alleles in patients
with primary Sjögren’s syndrome. Scand J Rheumatol (2002) 31:362–5.
doi:10.1080/030097402320817095
60. Slade JD, Luskin AT, Gewurz H, Kra SC, Kirsner JB, Zeitz HJ. Inherited
deciency of second component of complement and HLA haplotype A10,B18
associated with inammatory bowel disease. Ann Intern Med (1978) 88:796–8.
doi:10.7326/0003-4819-88-6-796
61. Freeman HJ. Hereditary angioneurotic edema and familial Crohn’s disease.
Can J Gastroenterol (2000) 14:337–9.
62. Farkas H, Gyeney L, Nemesanszky E, Kaldi G, Kukan F, Masszi I, et al.
Coincidence of hereditary angioedema (HAE) with Crohn’s disease. Immunol
Invest (1999) 28:43–53. doi:10.3109/08820139909022722
63. Kolho KL, Paakkanen R, Lepistö AL, Wennerstom A, Meri S, Lokki ML. Novel
associations between major histocompatibility complex and pediatric-onset
inammatory bowel disease. J Pediatr Gastroenterol Nutr (2015). doi:10.1097/
MPG.0000000000000984
64. Sandahl TD, Kelsen J, Dige A, Dahlerup JF, Agnholt J, Hvas CL, etal. e
lectin pathway of the complement system is downregulated in Crohn’s disease
patients who respond to anti-TNF-alpha therapy. J Crohns Colitis (2014)
8:521–8. doi:10.1016/j.crohns.2013.11.007
65. Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A,
etal. Intestinal DMBT1 expression is modulated by Crohn’s disease-associated
IL23R variants and by a DMBT1 variant which inuences binding of the tran-
scription factors CREB1 and ATF-2. PLoS One (2013) 8:e77773. doi:10.1371/
journal.pone.0077773
66. Kim TH, Lee SH, Lee HM, Jung HH, Lee SH, Cho WS, etal. Increased expres-
sion of glycoprotein 340 in the ethmoid sinus mucosa of patients with chronic
sinusitis. Arch Otolaryngol Head Neck Surg (2007) 133:1111–4. doi:10.1001/
archotol.133.11.1111
67. Renner M, Bergmann G, Krebs I, End C, Lyer S, Hilberg F, et al. DMBT1
confers mucosal protection in vivo and a deletion variant is associated
with Crohn’s disease. Gastroenterology (2007) 133:1499–509. doi:10.1053/j.
gastro.2007.08.007
68. Hamm CM, Reimers MA, McCullough CK, Gorbe EB, Lu J, Gu CC, etal.
NOD2 status and human ileal gene expression. Inamm Bowel Dis (2010)
16:1649–57. doi:10.1002/ibd.21208
69. Muller H, End C, Weiss C, Renner M, Bhandiwad A, Helmke BM, et al.
Respiratory deleted in malignant brain tumours 1 (DMBT1) levels increase
during lung maturation and infection. Clin Exp Immunol (2008) 151:123–9.
doi:10.1111/j.1365-2249.2007.03528.x
70. Madsen J, Sorensen GL, Nielsen O, Tornoe I, im L, Fenger C, etal. A vari-
ant form of the human deleted in malignant brain tumor 1 (DMBT1) gene
shows increased expression in inammatory bowel diseases and interacts
with dimeric trefoil factor 3 (TFF3). PLoS One (2013) 8:e64441. doi:10.1371/
journal.pone.0064441
71. Aamann L, Vestergaard EM, Gronbaek H. Trefoil factors in inammatory
bowel disease. World J Gastroenterol (2014) 20:3223–30. doi:10.3748/wjg.v20.
i12.3223
Conict of Interest Statement: e authors declare that the research was con-
ducted in the absence of any commercial or nancial relationships that could be
construed as a potential conict of interest.
Copyright © 2016 Reichhardt and Meri. is is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). e use,
distribution or reproduction in other forums is permitted, provided the original
author(s) or licensor are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms.