ArticlePDF Available

Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold


Abstract and Figures

The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.
Content may be subject to copyright.
Research Article
Structures of SALSA/DMBT1 SRCR domains reveal
the conserved ligand-binding mechanism of the ancient
SRCR fold
Martin P Reichhardt
, Vuokko Loimaranta
, Susan M Lea
, Steven Johnson
The scavenger receptor cysteine-rich (SRCR) family of proteins
comprises more than 20 membrane-associated and secreted
molecules. Characterised by the presence of one or more copies of
the ~110 amino-acid SRCR domain, this class of proteins have
widespread functions as antimicrobial molecules, scavenger re-
ceptors, and signalling receptors. Despite the high level of struc-
tural conservation of SRCR domains, no unifying mechanism for
ligand interaction has been described. The SRCR protein SALSA, also
known as DMBT1/gp340, is a key player in mucosal immunology.
Based on detailed structural data of SALSA SRCR domains 1 and 8,
we here reveal a novel universal ligand-binding mechanism for
SALSA ligands. The binding interface incorporates a dual cation-
binding site, which is highly conserved across the SRCR superfamily.
Along with the well-described cation dependency on most SRCR
domainligand interactions, our data suggest that the binding
mechanism described for the SALSA SRCR domains is applicable to
all SRCR domains. We thus propose to have identied in SALSA a
conserved functional mechanism for the SRCR class of proteins.
DOI 10.26508/lsa.201900502 | Received 26 July 2019 | Revised 14 February
2020 | Accepted 14 February 2020 | Published online 25 February 2020
The salivary scavenger and agglutinin (SALSA), also known as gp340,
deleted in malignant brain tumors 1(DMBT1) and salivary ag-
glutinin (SAG), is a multifunctional molecule found in high abun-
dance on human mucosal surfaces (1,2,3,4). SALSA has widespread
functions in innate immunity, inammation, epithelial homeosta-
sis, and tumour suppression (5,6,7). SALSA binds and agglutinates a
broad spectrum of pathogens including, but not limited to, human
immunodeciency virus type 1, Helicobacter pylori,Salmonella
enterica serovar Typhimurium, and many types of streptococci (8,9,
10,11). In addition to its microbial scavenging function, SALSA has
been suggested to interact with a wide array of endogenous im-
mune defence molecules. These include secretory IgA, surfactant
proteins A (SP-A) and D (SP-D), lactoferrin, mucin-5B, and com-
ponents of the complement system (1,2,12,13,14,15,16,17,18).
SALSA thus engages innate immune defence molecules and has
been suggested to cooperatively mediate microbial clearance and
maintenance of the integrity of the mucosal barrier.
The 300- to 400-kD SALSA glycoprotein is encoded by the DMBT1
gene. The canonical form of the gene encodes 13 highly conserved
scavenger receptor cysteine-rich (SRCR) domains, followed by two
C1r/C1s, urchin embryonic growth factor and bone morphogenetic
protein-1 (CUB) domains that surround a 14
SRCR domain, and
nally a zona pellucida domain at the C terminus (19,20). The rst 13
SRCRs are 109 aa domains found as pearls on a stringseparated
by SRCR-interspersed domains (SIDs) (Fig 1A)(1,21). The SIDs are 20-
to 23-aa-long stretches of predicted disorder containing a number
of glycosylation sites, which have been proposed to force them into
an extended conformation of roughly 7 nm (7). In addition to this
main form, alternative splicing and copy number variation mech-
anisms lead to expression of variants of SALSA containing variable
numbers of SRCR domains in the N-terminal region.
The SRCR protein superfamily include a range of secreted and
membrane-associated molecules, all containing one or more SRCR
domains. For a number of these molecules, the SRCR domains have
been directly implicated in ligand binding. These include CD6 sig-
nalling via CD166, CD163-mediated clearance of the haemoglobin
haptoglobin complex, Mac-2 binding proteins (M2bps) interaction
with matrix components, and the binding ofmicrobial ligands by the
scavenger receptors SR-A1, SPα, and MARCO (22,23,24,25,26,27).
Although the multiple SALSA SRCR domains likewise have been im-
plicated in ligand binding, the molecular basis for its diverse inter-
actions remains unknown.
To understand the multiple ligand-binding properties of the
SALSA molecule, we undertook an X-ray crystallographic study to
provide detailed information of the SALSA interaction surfaces. We
here provide the atomic resolution structures of SALSA SRCR do-
mains 1 and 8. We identify cation-binding sites and demonstrate
their importance for ligand binding. By comparing our data to
previously published structures of SRCR domains, we propose a
Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
Institute of Dentistry, University of Turku, Turku, Finland
Central Oxford Structural Molecular
Imaging Centre, University of Oxford, Oxford, UK
©2020Reichhardt et al. vol 3 | no 4 | e201900502 1of10
on 25 February, Downloaded from Online: 25 February, 2020 | Supp Info:
generalised binding mechanism for this ancient, evolutionarily
conserved, fold.
The scavenger receptor SALSA has a very wide range of described
ligands, including microbial, host innate immune, and ECM mole-
cules. To understand the very broad ligand-binding abilities of the
SALSA molecule, we applied a crystallographic approach to deter-
mine the structure of the ligand-binding SRCR domains of SALSA.
SRCR domains 1 and 8 (SRCR1 and SRCR8) were expressed in Dro-
sophila melanogaster Schneider S2 cells with a C-terminal His-tag.
The domains were puried by Ni-chelate and size-exclusion chro-
matography (SEC) and crystallised, and the structures were solved by
molecular replacement. This yielded the structures of SRCR1 and
SRCR8 at 1.77 and 1.29
A, respectively (Fig 1B). (For crystallographic
details, see Table 1).
The SALSA SRCRdomains reveal a classic globular SRCR-fold, with
four conserved disulphide bridges, as described for the SRCR type B
domains. The fold contains one α-helix and one additional single
helical turn. The N and C termini come together in a four-stranded
β-sheet. SALSA SRCR1-13 are highly conserved, with 88100% identity.
Variation is only observed in 9 of the 109 aa residues, all of these
observed in peripheral loops, without apparent structural signi-
cance. Combined, the data from SRCR1 and SRCR8 are thus valid
representations of all SALSA SRCR domains. Both SRCR1 and SRCR8
are stabilized by a metal ion buried in the globular fold. The
placement suggests the ion is bound during the folding of the do-
main and is modelled as Mg
, which is present in the original ex-
pression medium and in the crystallisation conditions of SRCR1.
So far, all described ligand-binding interactions of SALSA have
been shown to be Ca
-dependent. We therefore proceeded to ad-
dress the ligand-binding potential of the SRCR domains by adding
, and a cocktail of sugars to SRCR8 crystals before freezing.
This yielded a second crystal form ofSRCR8 with the original Mg
site 1, and two additional cations bound, sites 2 and 3 (Fig 2). All three
sites are class three cation-binding sites, with the coordination
obtained from residues in distant parts of the sequence (28).
Assignment of the identity of the ions at the paired site was carried
out by modelling Mg
or Ca
at each site, followed by renement of
the structure and analysis of the difference maps (Fig S1). These
revealed that Mg
best satised the data at both sites, consistent with
the 20-fold molar excess of Mg
over Ca
in the crystallisation so-
lution. However, it is worth noting that either site could likely ac-
commodate either cation depending on local concentration. Analysis
of the bond lengths and coordination numbers suggest that site 2 is a
canonical Mg
site, with octahedral geometry and average bond
lengths of 2.1
longer bond lengths, more consistent with a Ca
-binding site (29). The
at site 1 is coordinated by the backbone carbonyl groups of S1021
and V1060, as well as the side chains of D1023 and D1026, and two
waters, and is buried in the domain fold (Fig 2C). The Mg
at site 2 is
coordinated by D1019, D1020, and E1086 plus three waters (Fig 2D). The
at site 3 is coordinated by the side chains of D1020, D1058, D1059,
and N1081, with additional contributions from a water and an extra
density (Fig 2E). Attempts to model this extra density as any of the
sugars or alcohols present in the crystallisation solution failed to
produce a satisfactory t; therefore, it likely represents a superposition
of a number of molecules.
In contrast to the Mg
at site 1, these cations at sites 2 and 3 are
exposed on the surface of the domain, and the protein only con-
tributes a fraction of the coordination sphere, with the remainder
contributed by waters or small molecules from the crystallisation
solution. According to the literature, the majority of described SALSA
ligands are negatively charged. Thus, the surface-exposed cations
likely provide a mechanism for ligand binding for the SALSA SRCR
domains, whereby the anions of the ligand substitute for the waters
or the density at site 3 observed in our structure. To test this hy-
pothesis, site-directed mutagenesis was used, targeting the key
residues coordinating sites 2 and 3. Included in the further analysis
were single mutations D1019A and D1020A. While mutation of D1019 is
expected to only disrupt binding of cations at site 2, mutation of the
shared D1020, will likely affect binding of both cations.
As SALSA recognizes a very broad range of biological ligands, we
set out to test the effect of SRCR domain point mutations on in-
teractions with a wide array of biological ligands. These included
binding to (1) hydroxyapatite, a phosphate-rich mineral essential for
the binding of SALSA to the teeth surface, where it mediates anti-
microbial effects (30); (2) heparin, a sulphated glycosaminoglycan as
a mimic for theECM/cell surface, forwhich binding of SALSA hasbeen
described to affect cellular differentiation and microbial colonisation
(31); (3) Group A Streptococcus surface protein, Spy0843, a leucine-
rich repeat protein demonstrated to bind to SALSA (32)(Fig 3).
Figure 1. Crystal structure of SALSA domains SRCR1 and SRCR8.
(A) Schematic representation of the domain organization of full-length SALSA.
SRCR1 and SRCR8 are highlighted in green and blue, respectively. All SRCR
domains share >88% sequence identity. 100% identity is shared by SRCR3 and 7
(yellow) and SRCR10 and 11 (purple). (B) Front and side views of an overlay of
SRCR1 (green) and SRCR8 (blue), showing four conserved disulphide bridges
(yellow). Both SRCR1 and SRCR8 were found to coordinate a metal ion, modelled as
(dark green for SRCR1 and dark blue for SRCR8). The limited structural
variation observed between SRCR1 and SRCR8 (92% sequence identity) imply that
these are appropriate representations of all SALSA SRCR domains 113.
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 2of10
Different binding assays provide an understanding of a gener-
alised binding mechanism of SALSA SRCR domains. While the WT
SRCR domain bound to all three ligands, both of the cation-binding
site mutations, D1019A and D1020A, abolished binding. This is
consistent with the bound cations acting as a bridge for ligand
interaction and thus provides a mechanistic explanation for the
binding properties of the SALSA SRCR domains. In the literature,
SALSA ligand binding has been described as specically calcium
dependent. To verify this, we conducted binding assays in an
MgEGTA-containing buffer (Fig 3C). The exchange of magnesium for
calcium abolished ligand interactions, thus supporting a calcium-
specic mediation of binding. As mutation of site 2 alone (modelled
as Mg
in our structure) abolished binding, our data suggest that
may occupy site 2 under physiological conditions.
All known members of the SRCR superfamily share a very high
degree of identity, both at the sequence and structural levels. An
FFAS search (33) of the SRCR8 sequence showed highest similarities
to CD163 SRCR5 (score: 65.4, 46% identity), M2bp (score: 64.4, 54%
identity), neurotrypsin (score: 61.5, 50% identity), MARCO (score:
59.4, 50% identity), CD5 SRCR1 (score: 48.8, 26% identity), and CD6
SRCR2 (score: 45.6, 60% identity). Using the Dali server (34), searches
for the cation-binding SRCR8 soak structureidentied two top hits as
M2bp (pdbid: 1by2) and CD6 SRCR3 (pdbid: 5a2e). These were iden-
tied with respective Z-scores of 21.4 (r.m.s.d. of 1.1
residues aligned) and 20.4 (r.m.s.d. of 1.5
A with 109 of 109 residues
aligned). Despite the classical division of SRCR superfamily proteins into
groups A and B, based on the conserved three versus four cysteine
domain structures correlate closely to both group A and group B SRCR
superfamily domains (Fig 4A).
For members of the SRCR superfamily where the SRCR domain
directly partakes in ligand binding, both microbial and endogenous
protein ligands have been described. For MARCO, crystallographic
structures identied a cation-binding site exactly corresponding to
Table 1. Data collection and renement statistics (molecular replacement).
SRCR1 (pdbid: 6sa4) SRCR8 (pdbid: 6sa5) SRCR8soak (pdbid: 6san)
Data collection
Space group P 2
Cell dimensions
A) 36.77, 45.19, 69.37 32.82, 40.82, 62.99 27.24, 46.64, 93.63
α,β,γ(°) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 90.00, 97.37, 90.00
Resolution (
A) 28.521.77 (1.801.77)
40.821.29 (1.311.29) 46.641.36 (1.391.36)
0.17 (1.36) 0.117 (1.12) 0.074 (0.801)
I/σI8.1 (1.1) 8.6 (0.9) 14.9 (2.2)
Completeness (%) 99.8 (99.3) 100 (99.6) 98.3 (96.9)
Redundancy 6.3 (6.6) 11.4 (8.0) 6.6 (6.4)
Resolution (
A) 28.521.77 (1.951.77) 34.271.29 (1.351.29) 30.951.36 (1.391.36)
No. of reections 11,730 21,958 49,166
0.186/0.229 (0.266/0.348) 0.155/0.188 (0.326/0.279) 0.186/0.226 (0.267/0.326)
No. of atoms
Protein 824 829 3,192
Ligand/ion 26 18 34
Water 109 124 358
Protein 22.36 16.80 17.48
Ligand/ion 49.33 54.03 26.08
Water 31.15 33.66 35.55
R.m.s. deviations
Bond lengths (
A) 0.006 0.009 0.007
Bond angles (°) 0.809 1.026 0.87
Ramachandran outliers 0 0 0
Rotamer outliers 0 0 0
Number of crystals was one for each structure.
Values in parentheses are for highest resolution shell. Data from SRCR1 and SRCR8 crystals were collected on Diamond beamline I04, while data for SRCR8soak
were collected on beamline I03.
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 3of10
site 2 in the SALSA SRCR8 domain (26) and point mutations of this site
abolished function. Common to the MARCO and SALSA SRCR domains
is the cluster of negatively charged residues coordinating the
functionally important cations. A similar cluster is also observed in
the SRCR3 domain of CD6 and has been shown by mutagenesis to be
directly involved in binding to the human surface receptor CD166.
Indeed, a point mutation of D291A (corresponding to D1019 of SALSA-
SRCR8) reduced the ligand-binding potential of CD6 to less than 10%
(27). Furthermore, mutational studies of SRCR domains 2 and 3 from
CD163 proved an involvement of this specic site in the binding of the
haemoglobinhaptoglobin complex (36). All structural evidencefrom
mutational studies of SRCR domains thus indicate a conserved
surface-mediating ligand binding (Fig 4B).
Interestingly, various levels of calcium-dependency on ligand
interactions have been described for all SRCR domains directly
involved with binding. SR-A1, Spα,MARCO,CD5,andCD6allrelyon
calcium for interactions with microbial ligands (24,25,26,37,38,39).
Furthermore, the binding of CD163 to the haemoglobinhaptoglobin
complex is calcium-dependent, while CD6 also recognizes endoge-
nous surface structures (other than CD166) in a calcium-dependent
manner (40). This suggests that the cation-dependent binding
mechanism identied for SALSA is a general conserved feature of all
SRCR domains. Indeed, sequence alignment of SRCR domains from 10
different SRCR superfamily proteins, all with SRCR domains directly
involved with ligand binding, reveals a very high level of conservation
of the two cation-binding sites identied in the SALSA domains (Fig
4C). The ConSurf server is a tool to estimate (on a scale from 1 to 9) the
level of evolutionary conservation of residues in a given fold (41). A
search with the SRCR8 model shows that D1019, D1020, D1058, and
E1086 all score 7 (highly conserved), while N1081 and D1059 score 6
and 4, respectively (thus less conserved). Whenever sequence identity
is not conserved, substitutions are observed with other residues
overrepresented in cation-binding sites (D, E, Q, and N) (28,29). The
cation-binding sites identied in the SALSA SRCR domains, thus,
appear to be a highly conserved feature of the general SRCR fold.
Figure 2. Crystal structure of SRCR8 with bound magnesium ions indicates
mechanism of ligand binding.
(A) Surface charge distribution of SRCR8 (calculated without the presence of
cations) shows a positive cluster on one side with a strong negative cluster
on the other. The negative cluster expands across ~300
and mediates
the binding of three cations (green). (B) Representation of the residues
coordinating the three cations. The upper Mg
, site 1, sits somewhat buried
in the structure and may be essential for structural stability. The lower
cations at sites 2 and 3 are more exposed. (C) Detailed view of the
coordination of the upper Mg
, site 1. The coordination number of six is
achieved by two waters, two backbone carbonyls, and two side chain
carboxylates. (D) Detailed view of the coordination of the cation at site 2,
modelled as Mg
based on bond length and coordination number. Here, the
coordination number of six is achieved by three waters and three side
chain carboxylates. (E) Detailed view of the coordination of the cation at site
3, modelled as Mg
. The coordination is achieved by three side chain
carboxylates, one side chain amide, and an unmodelled density that is
assumed to be a superposition of crystallisation condition compounds.
Figure 3. Mutating the cation-binding residues of scavenger receptor cysteine-
rich (SRCR) domains abolish function.
Through multiple ligand-binding assays, we demonstrated the functional
importance of cation binding by the SRCR domains. Mutations affecting site 2
(D1019A) and mutations affecting sites 2 and 3 (D1020A) both abolish function.
(A) WT and mutant forms of SRCR8 were incubated with hydroxyapatite beads in a
-containing buffer. After extensive washing, bound protein was eluted with
EDTA. Eluted fractions were run on a 420% SDSPAGE gel and visualized by
Coomassie staining. Only WT SRCR8 bound hydroxyapatite. (B) WT and mutant
forms of SRCR8 were own over a heparin (HiTrap HP, 1 ml) column in a Ca
containing buffer. Protein bound to the column was eluted with 0.5 M EDTA.
Only WT SRCR8 bound the heparin column. Traces: SRCR8 (blue), D1020A (pink),
D1019A (red), conductivity (brown). (C) In an ELISA-based setup, a concentration
range of the Spy-2 domain of Spy0843 was coated (1100 μg/ml). WT and
mutant SRCR8 domains were added (100 μg/ml), and binding was detected with a
monoclonal anti-SALSA antibody. Binding was only observed for WT SRCR8. (D)
Overview of ligand-binding studies; + denotes binding, denotes no binding.
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 4of10
Figure 4. Conserved ligand-binding motif across scavenger receptor cysteine-rich (SRCR) domains.
SRCR domains from multiple proteins engage in cation-depende nt ligand interactions. (A) Structural overlay of domains from seven SRCR superfamily proteins, all with
ligand binding mediated through the SRCR domain. This reveals a highly conserved fold across both type A and type B SRCR domains. SRCR1 (pale green), SRCR8 (light
blue), MARCO (pdbid: 2oy3, sand), CD163 (pdbid: 5jfb, purple), CD5 (pdbid: 2OTT, grey), CD6 (pdbid: 5a2e, pink), M2bp (pdbid: 1by2, yellow), and murine neurotrypsin (pdbid:
6h8m, teal) (35). SALSA magnesium: green, MARCO magnesium: blue. (B) Surface representation of CD6 SRCR3, SALSA SRCR8, and MARCO in same orientation. Point
mutations with a veried impact on ligand binding are highlighted in red, indicating a conserved surface involved in ligand binding. Bound magnesium is highlighted in
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 5of10
Structural inspection of the overlay of SALSA SRCR8 with the corre-
sponding region in the other known SRCR domain structures clearly
shows the potential for cation binding at these sites. We thus propose
that the cation-binding sites identied here are an essential feature of
the ancient SRCR fold and are a conserved mechanism responsible for
mediating ligand binding in the class of SRCR superfamily proteins.
Although SALSA has previously been described to interact with a wide
range of biological ligands, little has been known of the binding
mechanisms. Furthermore, it has not been known if SALSA interacts with
various ligands in a similar way or if distinct binding sites are used. Here,
we demonstrate that mutations of a dual cation-binding site interrupt
interactions with a representative selection of very different types of
ligands. Specic disruption of site 2 was sufcient to abolish ligand
binding. We modelled the cation at site 2 in our crystal as Mg
, based on
an analysis of bond length, coordination number, and behaviour of
crystallographic renements with different cations modelled. However,
experimental data demonstrated that binding to the ligands tested was
only dependent on the presence of Ca
and not Mg
previous descriptions of most SALSAligand interactions (5,6,42). In the
extracellular compartment, the molar concentration of Ca
is higher
than Mg
(2.5 and 1 mM for calcium and magnesium, respectively), and
it is therefore likely that both sites 2 and 3 will be occupied by Ca
in a
physiological setting. The identication of this dual Ca
-binding site
thus provides an explanation for the Ca
dependency of all SALSA
ligand interactions described in the literature, suggesting this mech-
anism of binding is applicable to all SALSA SRCR ligands. Multiple
studies have proposed a role for the motif GRVEVxxxxxW in ligand
binding (43,44,45). The crystal data show that this peptide sequence is
binding, although mutations within this sequence are likely to perturb
the overall fold. This motif thus does not appear to have any physi-
ological relevance as dening a ligand-binding site.
The conserved usage of a single ligand-binding area for multiple
interactions suggests that each SALSA SRCR domain engages in one li-
gand interaction. A common feature of the ligands described here, as well
as a number of other ligands such as DNA and LPS, is the presence of
repetitive negatively charged motifs (31). We analysed ligand binding by
individual SRCR domains in surface-plasmon resonance and isothermal
calorimetry assays, but interactions were observed to be of very low
afnity, making reliable measurementsunfeasible.Thisisnotsurprising
for a molecule such as SALSA, where the molecular makeup with the full
extension of 13 repeated units, interspersed by predicted nonstructured
exible SIDs, provides a molecule that can generate high-avidity inter-
actions with repetitive ligands, despite having only low-afnity interac-
tions for an individual domain. Furthermore, it has been suggested that
SALSA in body secretions may oligomerize into larger complexes (5,46,47,
48), probably via the C-terminal CUB and zona pellucida domains. The
repetitive nature and possible oligomerization allow SALSA to not only
engage with a repetitive ligand on one surface (e.g., LPS or Spy0843 on
microbes) but also engage in multiple ligand interactions simultaneously.
This would be relevant for its interactions with other endogenous de-
fence molecules, such as IgA, SPs, and complement components, where a
cooperative effect on microbial clearance has been demonstrated (12,16,
17,49). In addition, this model of multiple ligand binding would be rel-
evant for microbes described to use SALSA for colonisation of the teeth or
the host epithelium (10,50,51)(Fig 5).
SALSA belongs to the SRCR superfamily, a family of proteins char-
acterised by the presence of one or more copies of the ancient and
evolutionarily highly conserved SRCR fold (52).Althoughacoupleof
SRCR domains, such as the ones found in complement factor I and
hepsin, have not been described to bind ligands directly, most others
have (53,54). SRCR superfamily members, such as SALSA, SR-A1, Spα,
SSc5D, MARCO, CD6, and CD163, have broad scavenger-receptor func-
tions, recognizing a broad range of microbial surface structures and
mediate clearance (24,25,26,37). Although this potentially is relevant for
all SRCR superfamily proteins, some members of the family have
With the exception of the CD6CD166 interactions, most described
SRCRligand interactions are calcium dependent, irrespective of the
ligand (24,25,26,37,38,39,40). A cation-binding site is conserved across
SRCR domains, and multiple studies support a role for this site in ligand
binding. Even the specialised CD6CD166 interaction uses the same
surface for binding, despite having lostthe calcium dependency (27).
Our studies have thus identied a dual cation-binding site as es-
sential for SALSAligand interactions. Analysis of SRCR folds from
various ligand-binding domains reveals a very high level of conser-
vationoftheresiduesatthisdualsite. The conservation of this site,
along with the well-described cation dependency on most SRCRligand
interactions, suggests that the binding mechanism described for the
SALSA SRCR domains is applicable to all SRCR domains. We thus
propose to have identied in SALSA a conserved functional mechanism
for the SRCR class of proteins. This notion is further supported by the
specic lack of conservation of these residues observed in the SRCR
domains of complement factor I and hepsin, where no ligand binding
has been shown. The SRCR domains in these two molecules may thus
represent an evolutionary diversion from the common broad ligand-
binding potential of the SRCR fold. The novel understanding of the SRCR
domain generated here will allow for an interesting future targeting of
other SRCR superfamily proteins, with the potential of modifying function.
Materials and Methods
Expression of recombinant proteins
Insect cell expression
Codon-optimized DNA (GeneArt; Thermo Fisher Scientic) was cloned
into a modied pExpreS2-2 vector (ExpreS2ion Biotechnologies) with a
green. (C) Clustal Omega (EMBL-EBI) sequence alignment of SRCR domains from 10 SRCR superfamily proteins. Conservation of the cation-binding sites are displayedingreen
(site 2) and purple (site 3). Dark colouring indicates 100% identity with the SALSA sites, and lighter colouring indicates conservation of residues commonly implicated in cation-
binding (D, E, Q, or N). Cysteines are highlightedin yellow, and over all sequence identity is denoted by *(100%), :(strongly similar chemical properties), and .(weakly similar chemical
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 6of10
C-terminal His-6 tag. The puried plasmid was transformed into S2
cells grown in EX-CELL 420 (Sigma-Aldrich) with 25 μl ExpreS2 Insect-TR
5X (ExpreS2ion Biotechnologies). Selection for stable cell lines (4 mg/
ml geneticin [Thermo Fisher Scientic]) and expansion were carried
out according to the manufacturers instructions.
Escherichia coli expression
DNA strings (GeneArt; Thermo Fisher Scientic) were cloned into
pETM-14 and transformed into M15pRep cells. Protein expression was
carried out in LB media (with 30 μg/ml kanamycin). Cells were in-
duced with 1 mM IPTG. The cultures were centrifuged (3,220g,15min)
and the cell pellets resuspended and lysed in PBS containing 1 mg/
ml DNase and 1 mg/ml lysozyme.
Protein purication
SRCR domains
Insect culture supernatant was collected by centrifugation (1,000gat
30 min), ltered and loaded onto a Roche cOmplete Ni
tography column (1 ml, Cat. no. 06781543001; Sigma-Aldrich), washed
in 20 CV buffer (50 mM Tris, pH: 9.0, 200 mM NaCl). Bound protein was
eluted with 250 mM imidazole. Following this, SEC was carried out on
a Superdex 75 16/60 HR column (GE Healthcare) equilibrated in 10
mM Tris, pH: 7.5, 200 mM NaCl.
Lysed cell pellets were homogenized and centrifuged at 20,000gfor 30
min. The ltered supernatant was loaded onto a Ni
200 mM NaCl, 20 mM imidazole). Bound protein was eluted (in 50 mM
Tris, pH: 8.5, 200 mM NaCl, 250 mM imidazole), concentrated, and
subjected to SEC (Superdex 75 16/60 HR column; GE Healthcare).
Crystallisation, X-ray data collection, and structure
Puried SRCR1 and SRCR8 were concentrated to 20 mg/ml. SRCR1 was
hexahydrate, 10% (wt/vol) PEG8000, 0.1 M Tris, pH: 7.0, and crystallised
in 400 nl drops by the vapor diffusion method at 21°C. SRCR8 was
mixed with an equal volume of mother liquor containing 0.1 M LiSO
20% (wt/vol) PEG6000, 0.01 M Hepes, pH: 6.5, and crystallised in 800 nl
drops. For SRCR8 + cation crystals were grown in 0.2 M MgCl
hydrate, 20% (vol/vol) isopropanol, 0.1 M Hepes, pH: 7.5, and crys-
tallised in 400 nl drops. The crystallisation buffer was supplemented
with 10 mM Mg
and 10 mM Ca
, as well as 10 mM maltose, D-ga-
lactose, D-saccharose, D-mannose, D-glucose, and sucrose octa-
sulphate (all Sigma-Aldrich), 24 h prior to freezing. All crystals were
cryoprotected in mother liquor supplemented with 30% glycerol and
. Data were collected at a temperature of 80 K
Figure 5. SALSA scavenger receptor cysteine-rich (SRCR) cation-binding motif
reveals a conserved mechanism for broad-spectrum ligand interactions of
SRCR superfamily molecules.
Based on mutational studies and structural information across SRCR proteins, we
propose a generalised mechanism of ligand interaction mediated by the
cation-binding surface motif of the evolutionarily ancient SRCR fold (left side).
SALSA has been described to bind a broad range of ligands, incorporating into a
complex network of binding partners on the body surfaces and the colonizing
microbiota. The multiple SRCR domains of full-length SALSA bind repetitive
targets (both protein and carbohydrate structures) on the surface of microbes.
The secreted uid-phase molecule may thus lead to microbial agglutination
and clearance. However, the repetitive form of binding sites will allow for
simultaneous binding to endogenous targets as well. This being, for example, 1)
binding of IgA, collectins, and complement components to induce a
cooperative antimicrobial effect; 2) binding of hydroxyapatite on the tooth
surface; and 3) ECM proteins and glycosaminoglycans, as well as mucus
components of the epithelial surface (such as heparin, galectin 3, and mucins)
(right side). The cation-binding motif described in SALSA is conserved in most
other SRCR proteins. For CD6, CD163, and MARCO, mutational studies support a
crucial role for this area in ligand interactions. CD163 binds the
haemoglobinhaptoglobin complex and microbial surfaces. CD6 binds
endogenous ligands but also engages in microbial binding. MARCO forms
multimers and binds microbial surface structures. Other SRCR proteins with
similar functions and conserved cation sites include SR-A1, Sp-α, SSc5D, and
M2bp. The functional role of the neurotrypsin SRCR domains is not known. The
remarkable repetitive formation of multiple SRCR domains in many SRCR
superfamily proteins, with several domains containing a binding site with a broad
specicity, would supposedly allow for interactions with multiple ligands
simultaneously. The SRCR fold thus appears to be an important functional
component of scavenging molecules engaging in complex network of
interactions. The multiple SRCR domains shown for SALSA, CD163, and
neurotrypsin are represented as copies of protein-specic SRCR domains with
known structure. Conserved cation-coordinating residues are highlighted in red.
SRCR8soak (light blue), MARCO (pdbid: 2oy3, sand), CD163 (pdbid: 5jfb, purple),
CD6 (pdbid: 5a2e, grey), M2bp (pdbid: 1by2, yellow), and murine neurotrypsin
(pdbid: 6h8m, teal).
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 7of10
on beamlines I04, at a wavelength of 1.0718
A (for SRCR1 and SRCR8)
and I03, at a wavelength of 0.9762
A (for SRCR8cat) at the Diamond
Light Source, as specied in Table 1. The structure of SRCR8 was
solved by molecular replacement using MolRep within CCP4 (56) with
the structure of CD6 SRCR domain 3 (PDB ID 5a2e (27)). The structures
of SRCR1 and SRCR8 soaked in cations were solved by molecular
replacement using the structure of SRCR8. Renement and re-
building were carried out in Phenix and Coot (57,58). Assignment of
metal ions was carried out by rst rening the structure without
anything in the metal binding sites, followed by addition of com-
binations of probable ligands, and re-renement in phenix.rene
using restraints generated by phenix.ready_set for each combina-
tion. The structures were characterised by the statistics shown in
Table 1 with no Ramachandran outliers. Protein structure gures
were prepared using Pymol version 2.0 (Schr¨
odinger, LLC).
Hydroxyapatite binding assay
150 μl hydroxyapatite nanoparticle suspension (Cat. no. 702153;
Sigma-Aldrich) was washed into buffer (10 mM Hepes, pH: 7.5, 150 mM
NaCl, 1 mM Ca
). Beads were incubated in 80 μl SRCR8, SRCR8 D34A,
or SRCR8 D35A(all at 0.5 mg/ml inthe same buffer) with shaking for 1
h at RT. Beads were spun and washed 6× in 1 ml buffer. Bound protein
was eluted in 100 μl 0.5 M EDTA and visualized by SDSPAGE (420%;
Bio-Rad) and Coomassie staining (Instant Blue, Expedeon).
Heparin binding assay
SRCR8, SRCR8 D34A, or SRCR8 D35A in 10 mM Hepes, pH: 7.5, 10 mM
NaCl, 1 mM Ca
were loaded onto a HiTrap Heparin HP column (1 ml;
GE Healthcare), equilibrated in the same buffer. Bound protein was
then eluted with 10 mM Hepes, pH: 7.5, 10 mM NaCl, 20 mM EDTA.
Spy-2 binding assay
On a MaxiSorp plate (Nunc), 100 μl puried Spy-2 was coated O/N at
4°C in a concentration ranging from 0.032 to 3.2 μM in coating buffer
(100 mM NaHCO
buffer, pH: 9.5). The plate was blocked in 1% gelatine
in PBS, and SRCR8, SRCR8 D34A, and SRCR8 D35A were added (all at 7.1
μM in 10 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM Ca
, 0.05% Tween20).
diluted 1:10,000 (1G4; Novus Biologicals) and HRP-conjugated rabbit
anti-mouse antibody 1:10,000 (W4028; Promega). The plate was de-
velopedwith2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(Sigma-Aldrich) and analysed by spectrophotometry at 405 nm. To test
calcium-specic dependency of the interaction, the WT assay above
was repeated in a buffer containing 10 mM Hepes, pH 7.5, 150 mM NaCl,
Data availability
Structure factors and coordinates from this publication have been
deposited to the PDB database and assigned
the identiers: SRCR1 pdbid: 6sa4, SRCR8 pdbid: 6sa5,SRCR8soakwith
three cations pdbid: 6san.
Supplementary Information
Supplementary Information is available at
We acknowledge Diamond Light Source and the staff of beamlines I03 and I04
for access under proposal MX18069. The Central Oxford Structural Molecular
and Imaging Centre is supported by the Wellcome Trust (201536). MP Reich-
hardt was nancially supported by grants from the Wihuri Foundation and the
Finnish Cultural Foundation. Staff andexperimental costs in SM Lealaboratory
were supported by a Wellcome Investigator Award (100298) and an Medical
Research Council (UK) programme grant (M011984). V Loimaranta was sup-
ported by the Turku University Foundation.
Author Contributions
MP Reichhardt: conceptualization, data curation, formal analysis,
funding acquisition, investigation, and writingoriginal draft, review,
and editing.
V Loimaranta: resources.
SM Lea: conceptualization, data curation, formal analysis, funding ac-
quisition, validation, investigation, methodology, project administration,
and writingreview and editing.
S Johnson: conceptualization, data curation, formal analysis, super-
vision, validation, investigation, visualization, methodology, project
administration, and writingoriginal draft, review, and editing.
Conict of Interest Statement
The authors declare that they have no conict of interest.
1. Holmskov U, Lawson P, Teisner B, Tornoe I, Willis AC, Morgan C, Koch C,
Reid KB (1997) Isolation and characterization of a new member of the
scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung
surfactant protein-D binding molecule. J Biol Chem 272: 1374313749.
2. Thornton DJ, Davies JR, Kirkham S, Gautrey A, Khan N, Richardson PS,
Sheehan JK (2001) Identication of a nonmucin glycoprotein (gp-340)
from a puried respiratory mucin preparation: Evidence for an
association involving the MUC5B mucin. Glycobiology 11: 969977.
3. Schulz BL, Oxley D, Packer NH, Karlsson NG (2002) Identication of two
highly sialylated human tear-uid DMBT1 isoforms: The major high-
molecular-mass glycoproteins in human tears. Biochem J 366: 511520.
4. Reichhardt MP, Jarva H, de Been M, Rodriguez JM, Jimenez Quintana E,
Loimaranta V, de Vos WM, Meri S (2014) The salivary scavenger and
agglutinin in early life: Diverse roles in amniotic uid and in the infant
intestine. J Immunol 193: 52405248. doi:10.4049/jimmunol.1401631
5. Madsen J, Mollenhauer J, Holmskov U (2010) Review: Gp-340/DMBT1 in
mucosal innate immunity. Innate Immun 16: 160167. doi:10.1177/
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 8of10
6. Reichhardt MP, Meri S (2016) SALSA: A regulator of the early steps of
complement activation on mucosal surfaces. Front Immunol 7: 85.
7. Reichhardt MP, Holmskov U, Meri S (2017) SALSA-A dance on a slippery
oor with changing partners. Mol Immunol 89: 100110. doi:10.1016/
8. Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I,
Frangsmyr L, Holmskov U, Lefer H, Nilsson C, et al (2000) Salivary
agglutinin, which binds Streptococcus mutans and Helicobacter pylori,
is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem
275: 3986039866. doi:10.1074/jbc.m006928200
9. Hartshorn KL, White MR, Mogues T, Ligtenberg T, Crouch E, Holmskov U
(2003) Lung and salivary scavenger receptor glycoprotein-340
contribute to the host defense against inuenza A viruses. Am J Physiol
Lung Cell Mol Physiol 285: L1066L1076. doi:10.1152/ajplung.00057.2003
10. Loimaranta V, Jakubovics NS, Hytonen J, Finne J, Jenkinson HF, Stromberg
N (2005) Fluid- or surface-phase human salivary scavenger protein
gp340 exposes different bacterial recognition properties. Infect Immun
73: 22452252. doi:10.1128/iai.73.4.2245-2252.2005
11. Rosenstiel P, Sina C, End C, Renner M, Lyer S, Till A, Hellmig S, Nikolaus S,
Folsch UR, Helmke B, et al (2007) Regulation of DMBT1 via NOD2 and TLR4
in intestinal epithelial cells modulates bacterial recognition and
invasion. J Immunol 178: 82038211. doi:10.4049/jimmunol.178.12.8203
12. Rundegren J, Arnold RR (1987) Differentiation and interaction of
secretory immunoglobulin A and a calcium-dependent parotid
agglutinin for several bacterial strains. Infect Immun 55: 288292.
13. Boackle RJ, Connor MH, Vesely J (1993) High molecular weight non-
immunoglobulin salivary agglutinins (NIA) bind C1Q globular heads and
have the potential to activate the rst complement component. Mol
Immunol 30: 309319. doi:10.1016/0161-5890(93)90059-k
14. Tino MJ, Wright JR (1999) 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 20: 759768. doi:10.1165/
15. Oho T, Bikker FJ, Nieuw Amerongen AV, Groenink J (2004) A peptide
domain of bovine milk lactoferrin inhibits the interaction between
streptococcal surface protein antigen and a salivary agglutinin peptide
domain. Infect Immun 72: 61816184. doi:10.1128/iai.72.10.6181-6184.2004
16. Leito JT, Ligtenberg AJ, van Houdt M, van den Berg TK, Wouters D (2011)
The bacteria binding glycoprotein salivary agglutinin (SAG/gp340)
activates complement via the lectin pathway. Mol Immunol 49: 185190.
17. Reichhardt MP, Loimaranta V, Thiel S, Finne J, Meri S, Jarva H (2012) The
salivary scavenger and agglutinin binds MBL and regulates the lectin
pathway of complement in solution and on surfaces. Front Immunol 3:
205. doi:10.3389/mmu.2012.00205
18. Madsen J, Sorensen GL, Nielsen O, Tornoe I, Thim L, Fenger C,
Mollenhauer J, Holmskov U (2013) A variant form of the human deleted in
malignant brain tumor 1 (DMBT1) gene shows increased expression in
inammatory bowel diseases and interacts with dimeric trefoil factor 3
(TFF3). PLoS One 8: e64441. doi:10.1371/journal.pone.0064441
19. Holmskov U, Mollenhauer J, Madsen J, Vitved L, Gronlund J, Tornoe I,
Kliem A, Reid KB, Poustka A, Skjodt K (1999) Cloning of gp-340, a putative
opsonin receptor for lung surfactant protein D. Proc Natl Acad Sci U S A
96: 1079410799. doi:10.1073/pnas.96.19.10794
20. Mollenhauer J, Holmskov U, Wiemann S, Krebs I, Herbertz S, Madsen J,
Kioschis P, Coy JF, Poustka A (1999) The genomic structure of the DMBT1
gene: Evidence for a region with susceptibility to genomic instability.
Oncogene 18: 62336240. doi:10.1038/sj.onc.1203071
21. Mollenhauer J, Wiemann S, Scheurlen W, Korn B, Hayashi Y, Wilgenbus
KK, von Deimling A, Poustka A (1997) DMBT1, a new member of the SRCR
superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain
tumours. Nat Genet 17: 3239. doi:10.1038/ng0997-32
22. Inohara H, Akahani S, Koths K, Raz A (1996) Interactions between
galectin-3 and Mac-2-binding protein mediate cell-cell adhesion.
Cancer Res 56: 45304534.
23. Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman HJ, Law SK,
Moestrup SK (2001) Identication of the haemoglobin scavenger
receptor. Nature 409: 198201. doi:10.1038/35051594
24. Santiago-Garcia J, Kodama T, Pitas RE (2003) The class A scavenger
receptor binds to proteoglycans and mediates adhesion of
macrophages to the extracellular matrix. J Biol Chem 278: 69426946.
25. Sarrias MR, Rosello S, Sanchez-Barbero F, Sierra JM, Vila J, Yelamos J, Vives J,
Casals C, Lozano F (2005) A role for human Sp alpha as a pattern recognition
receptor. JBiolChem280: 3539135398. doi:10.1074/jbc.m505042200
26. Ojala JR, Pikkarainen T, Tuuttila A, Sandalova T, Tryggvason K (2007)
Crystal structure of the cysteine-rich domain of scavenger receptor
MARCO reveals the presence of a basic and an acidic cluster that both
contribute to ligand recognition. J Biol Chem 282: 1665416666.
27. Chappell PE, Garner LI, Yan J, Metcalfe C, Hatherley D, Johnson S,
Robinson CV, Lea SM, Brown MH (2015) Structures of CD6 and its ligand
CD166 give insight into their interaction. Structure 23: 14261436.
28. Pidcock E, Moore GR (2001) Structural characteristics of protein binding
sites for calcium and lanthanide ions. J Biol Inorg Chem 6: 479489.
29. Dokmanic I, Sikic M, Tomic S (2008) Metals in proteins: Correlation
between the metal-ion type, coordination number and the amino-acid
residues involved in the coordination. Acta Crystallogr D Biol Crystallogr
64: 257263. doi:10.1107/S090744490706595X
30. Haukioja A, Loimaranta V, Tenovuo J (2008) Probiotic bacteria affect the
composition of salivary pellicle and streptococcal adhesion in vitro.
Oral Microbiol Immunol 23: 336343. doi:10.1111/j.1399-302x.2008.00435.x
31. End C, Bikker F, Renner M, Bergmann G, Lyer S, Blaich S, Hudler M, Helmke
B, Gassler N, Autschbach F, et al (2009) DMBT1 functions as pattern-
recognition molecule for poly-sulfated and poly-phosphorylated
ligands. Eur J Immunol 39: 833842. doi:10.1002/eji.200838689
32. Loimaranta V, Hytonen J, Pulliainen AT, Sharma A, Tenovuo J, Stromberg
N, Finne J (2009) Leucine-rich repeats of bacterial surface proteins serve
as common pattern recognition motifs of human scavenger receptor
gp340. J Biol Chem 284: 1861418623. doi:10.1074/jbc.m900581200
33. Jaroszewski L, Rychlewski L, Li Z, Li W, Godzik A (2005) FFAS03: A server for
proleprole sequence alignments. Nucleic Acids Res 33: W284W288.
34. Holm L, Laakso LM (2016) Dali server update. Nucleic Acids Res 44:
W351W355. doi:10.1093/nar/gkw357
35. Canciani A, Catucci G, Forneris F (2019) Structural characterization of the
third scavenger receptor cysteine-rich domain of murine neurotrypsin.
Protein Sci 28: 746755. doi:10.1002/pro.3587
36. Nielsen MJ, Andersen CB, Moestrup SK (2013) CD163 binding to
haptoglobin-hemoglobin complexes involves a dual-point electrostatic
receptor-ligand pairing. J Biol Chem 288: 1883418841. doi:10.1074/
37. Sarrias MR, Farnos M, Mota R, Sanchez-Barbero F, Ibanez A, Gimferrer I,
Vera J, Fenutria R, Casals C, Yelamos J, et al (2007) CD6 binds to pathogen-
associated molecular patterns and protects from LPS-induced septic
shock. Proc Natl Acad Sci U S A 104: 1172411729. doi:10.1073/
38. Vera J, Fenutria R, Canadas O, Figueras M, Mota R, Sarrias MR, Williams DL,
Casals C, Yelamos J, Lozano F (2009) The CD5 ectodomain interacts with
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 9of10
conserved fungal cell wall components and protects from zymosan-
induced septic shock-like syndrome. Proc Natl Acad Sci U S A 106:
15061511. doi:10.1073/pnas.0805846106
39. Nonaka M, Ma BY, Imaeda H, Kawabe K, Kawasaki N, Hodohara K,
Kawasaki N, Andoh A, Fujiyama Y, Kawasaki T (2011) Dendritic cell-
specic intercellular adhesion molecule 3-grabbing non-integrin (DC-
SIGN) recognizes a novel ligand, Mac-2-binding protein,
characteristically expressed on human colorectal carcinomas. J Biol
Chem 286: 2240322413. doi:10.1074/jbc.m110.215301
40. Patel DD, Wee SF, Whichard LP, Bowen MA, Pesando JM, Aruffo A, Haynes
BF (1995) Identication and characterization of a 100-kD ligand for CD6
on human thymic epithelial cells. J Exp Med 181: 15631568. doi:10.1084/
41. Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, Ben-Tal N
(2016) ConSurf 2016: An improved methodology to estimate and visualize
evolutionary conservation in macromolecules. Nucleic Acids Res 44:
W344W350. doi:10.1093/nar/gkw408
42. Ligtenberg AJ, Karlsson NG, Veerman EC (2010) Deleted in malignant
brain tumors-1 protein (DMBT1): A pattern recognition receptor with
multiple binding sites. Int J Mol Sci 11: 52125233. doi:10.3390/ijms1112521
43. Bikker FJ, Ligtenberg AJ, Nazmi K, Veerman EC, vant Hof W, Bolscher JG,
Poustka A, Nieuw Amerongen AV, Mollenhauer J (2002) Identication of
the bacteria-binding peptide domain on salivary agglutinin (gp-340/
DMBT1), a member of the scavenger receptor cysteine-rich superfamily. J
Biol Chem 277: 3210932115. doi:10.1074/jbc.m203788200
44. Bikker FJ, Ligtenberg AJ, End C, Renner M, Blaich S, Lyer S, Wittig R, vant
Hof W, Veerman EC, Nazmi K, et al (2004) Bacteria binding by DMBT1/
SAG/gp-340 is conned to the VEVLXXXXW motif in its scavenger
receptor cysteine-rich domains. J Biol Chem 279: 4769947703.
45. Leito JT, Ligtenberg AJ, Nazmi K, de Blieck-Hogervorst JM, Veerman EC,
Nieuw Amerongen AV (2008) A common binding motif for various
bacteria of the bacteria-binding peptide SRCRP2 of DMBT1/gp-340/
salivary agglutinin. Biol Chem 389: 11931200. doi:10.1515/bc.2008.135
46. Young A, Rykke M, Smistad G, Rolla G (1997) On the role of human salivary
micelle-like globules in bacterial agglutination. Eur J Oral Sci 105:
485494. doi:10.1111/j.1600-0722.1997.tb00235.x
47. Oho T, Yu H, Yamashita Y, Koga T (1998) Binding of salivary glycoprotein-
secretory immunoglobulin A complex to the surface protein antigen of
Streptococcus mutans. Infect Immun 66: 115121. doi:10.1128/iai.66.1.115-
48. Crouch EC (2000) Surfactant protein-D and pulmonary host defense.
Respir Res 1: 93108. doi:10.1186/rr19
49. White MR, Crouch E, van Eijk M, Hartshorn M, Pemberton L, Tornoe I,
Holmskov U, Hartshorn KL (2005) Cooperative anti-inuenza activities of
respiratory innate immune proteins and neuraminidase inhibitor. Am J
Physiol Lung Cell Mol Physiol 288: L831L840. doi:10.1152/
50. Jonasson A, Eriksson C, Jenkinson HF, Kallestal C, Johansson I, Stromberg
N (2007) Innate immunity glycoprotein gp-340 variants may modulate
human susceptibility to dental caries. BMC Infect Dis 7: 57. doi:10.1186/
51. Stoddard E, Cannon G, Ni H, Kariko K, Capodici J, Malamud D, Weissman D
(2007) gp340 expressed on human genital epithelia binds HIV-1
envelope protein and facilitates viral transmission. J Immunol 179:
31263132. doi:10.4049/jimmunol.179.5.3126
52. Sarrias MR, Gronlund J, Padilla O, Madsen J, Holmskov U, Lozano F (2004)
The scavenger receptor cysteine-rich (SRCR) domain: An ancient and
highly conserved protein module of the innate immune system. Crit Rev
Immunol 24: 137. doi:10.1615/critrevimmunol.v24.i1.10
53. Roversi P, Johnson S, Caesar JJ, McLean F, Leath KJ, Tsiftsoglou SA, Morgan
BP, Harris CL, Sim RB, Lea SM (2011) Structural basis for complement
factor I control and its disease-associated sequence polymorphisms.
Proc Natl Acad Sci U S A 108: 1283912844. doi:10.1073/pnas.1102167108
54. Koschubs T, Dengl S, Durr H, Kaluza K, Georges G, Hartl C, Jennewein S,
Lanzendorfer M, Auer J, Stern A, et al (2012) Allosteric antibody inhib ition
of human hepsin protease. Biochem J 442: 483494. doi:10.1042/
55. Burgueno-Bucio E, Mier-Aguilar CA, Soldevila G (2019) The multiple faces
of CD5. J Leukoc Biol 105: 891904. doi:10.1002/JLB.MR0618-226R
56. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan
RM, Krissinel EB, Leslie AG, McCoy A, et al (2011) Overview of the CCP4
suite and current developments. Acta Crystallogr D Biol Crystallogr 67:
235242. doi:10.1107/s0907444910045749
57. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and
development of Coot. Acta Crystallogr D Biol Crystallogr 66: 486501.
58. Adams PD, Baker D, Brunger AT, Das R, DiMaio F, Read RJ, Richardson DC,
Richardson JS, Terwilliger TC (2013) Advances, interactions, and future
developments in the CNS, Phenix, and Rosetta structural biology
software systems. Annu Rev Biophys 42: 265287. doi:10.1146/annurev-
License: This article is available under a Creative
Commons License (Attribution 4.0 International, as
described at
SALSA domain structures Reichhardt et al. vol 3 | no 4 | e201900502 10 of 10
... SAG is also known as gp340 and is expressed from the "deleted in membrane protein 1" gene (DMBT1). It is a large extracellular matrix protein which features 13 repeats of the scavenger receptor cysteine rich domain 1 (iSRCR; Reichhardt et al., 2020). GbpC has been shown to interact with these iSRCR domains in a calcium-dependent fashion (Purushotham and Deivanayagam, 2014;Mieher et al., 2018), but since the fold of iSRCR is in itself calcium-dependent (Reichhardt et al., 2020) it is unknown whether or not the calcium-binding site of the polymer adhesin domain is involved in this interaction. ...
... It is a large extracellular matrix protein which features 13 repeats of the scavenger receptor cysteine rich domain 1 (iSRCR; Reichhardt et al., 2020). GbpC has been shown to interact with these iSRCR domains in a calcium-dependent fashion (Purushotham and Deivanayagam, 2014;Mieher et al., 2018), but since the fold of iSRCR is in itself calcium-dependent (Reichhardt et al., 2020) it is unknown whether or not the calcium-binding site of the polymer adhesin domain is involved in this interaction. Both the interaction to the entire SAG and the individual iSRCR domains are inhibited by the addition of dextran, which could indicate that they compete for the same binding site. ...
Full-text available
Surface proteins in Gram-positive bacteria are often involved in biofilm formation, host-cell interactions, and surface attachment. Here we review a protein module found in surface proteins that are often encoded on various mobile genetic elements like conjugative plasmids. This module binds to different types of polymers like DNA, lipoteichoic acid and glucans, and is here termed polymer adhesin domain. We analyze all proteins that contain a polymer adhesin domain and classify the proteins into distinct classes based on phylogenetic and protein domain analysis. Protein function and ligand binding show class specificity, information that will be useful in determining the function of the large number of so far uncharacterized proteins containing a polymer adhesin domain.
... The functional role of LOXL2's SRCR domains has not yet been well characterized. It is assumed that they could be involved in protein-protein interactions by analogy to the function of the SRCR domains present in other members of the scavenger receptor superfamily [5,6]. Nevertheless, it has been recently observed that specific LOXL2 metastatic burden in the lungs and liver [34]. ...
Full-text available
Lysyl oxidase-like 2 (LOXL2) was initially described as an extracellular enzyme involved in extracellular matrix remodeling. Nevertheless, numerous recent reports have implicated intracellular LOXL2 in a wide variety of processes that impact on gene transcription, development, differentiation, proliferation, migration, cell adhesion, and angiogenesis, suggesting multiple different functions for this protein. In addition, increasing knowledge about LOXL2 points to a role in several types of human cancer. Moreover, LOXL2 is able to induce the epithelial-to-mesenchymal transition (EMT) process—the first step in the metastatic cascade. To uncover the underlying mechanisms of the great variety of functions of intracellular LOXL2, we carried out an analysis of LOXL2’s nuclear interactome. This study reveals the interaction of LOXL2 with numerous RNA-binding proteins (RBPs) involved in several aspects of RNA metabolism. Gene expression profile analysis of cells silenced for LOXL2, combined with in silico identification of RBPs’ targets, points to six RBPs as candidates to be substrates of LOXL2’s action, and that deserve a more mechanistic analysis in the future. The results presented here allow us to hypothesize novel LOXL2 functions that might help to comprehend its multifaceted role in the tumorigenic process.
... The latter contains three domains: a low density lipoprotein receptor type A (LDLRA), a class A scavenger receptor cysteine-rich (SRCR) and the C-terminal canonical serine protease domain (PDTMP) [102,104] (Figure 3). Each of LDLRA and SRCR domains, collectively called the stem region which contributes to substrate recognition as well as protein-protein interactions and ligand binding, harbor Ca 2+ binding sites [102,105,106]. PDTMP cleaves a variety of peptide substrates at Arginine and Lysine residues [106] which in the case of ACE2 occurs at the cluster of these two amino acids located in residues 697-716, noticing that this proteolysis is required for TMPRSS2-mediated cathepsin-independent virus entrance to the host cell [107]. ...
Full-text available
Since the spread of the deadly virus SARS-CoV-2 in late 2019, researchers have restlessly sought to unravel how the virus enters the host cells. Some proteins on each side of the interaction between the virus and the host cells are involved as the major contributors to this process: (1) the nano-machine spike protein on behalf of the virus, (2) angiotensin converting enzyme II, the mono-carboxypeptidase and the key component of renin angiotensin system on behalf of the host cell, (3) some host proteases and proteins exploited by SARS-CoV-2. In this review, the complex process of SARS-CoV-2 entrance into the host cells with the contribution of the involved host proteins as well as the sequential conformational changes in the spike protein tending to increase the probability of complexification of the latter with angiotensin converting enzyme II, the receptor of the virus on the host cells, are discussed. Moreover, the release of the catalytic ectodomain of angiotensin converting enzyme II as its soluble form in the extracellular space and its positive or negative impact on the infectivity of the virus are considered.
... TMPRSS2 as a type II transmembrane protease is composed of an intracellular N-terminus, a single pass transmembrane domain and an extracellular segment containing three domains: a low density lipoprotein receptor type A (LDLRA), a class A scavenger receptor cysteine-rich (SRCR) and the C-terminal canonical serine protease domain (PDTMP) [96,98]. Each of LDLRA and SRCR domains, collectively called the stem region which contributes to substrate recognition as well as protein-protein interactions and ligand binding, harbors Ca +2 binding sites [96,99,100]. PDTMP cleaves a variety of peptide substrates at Arginine and Lysine residues [100] which in the case of ACE2 occurs at the cluster of these two amino acids located in residues 697-716, noticing that this proteolysis is required for TMPRSS2-mediated cathepsin-independent virus entrance to the host cell [101]. ...
Full-text available
Since the spread of the deadly virus SARS-CoV2 in late 2019, researchers have restlessly been seeking for unraveling how the virus factually enters the host cells. Some proteins on each side of the interaction between the virus and the host cells are involved as the major contributors to this process: 1- the nano-machine Spike protein on behalf of the virus, 2- angiotensin converting enzyme II, the mono-carboxypeptidase and the key component of renin angiotensin system on behalf of the host cell, 3- some host proteases and proteins exploited by SARS-CoV2, In this review, the complex process of SARS-CoV2 entrance into the host cells with the contribution of the involved host proteins as well as the sequential conformational changes in the Spike protein tending to increase the probability of complexification of the latter with angiotensin converting enzyme II, the receptor of the virus on the host cells, are discussed. Besides, the release of the catalytic ectodomain of angiotensin converting enzyme II as its soluble form in the extracellular space and its positive or negative impact on the infectivity of the virus are considered.
... Our combined results on Ca 2+ dependent modulation of enzymatic activity and thermal stabilization of NT-mini unambiguously indicate the presence of a high-affinity Ca 2+ binding site(s) within the SP domain complemented by those likely to be found in the preceding SRCR domains [24,[61][62][63]. This is supported by the significantly different Ca 2+ concentrations we have found to be effective in stabilizing or activating NT-mini. ...
Full-text available
Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called “NT-mini” and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT’s heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.
... The postulated effects of SALSA involve multiple mechanisms including alterations in bacterial conformation, interactions with binding partners such as immune defense proteins, as well as other uncharacterized effects [10]. Whether reduced phagocytosis results from altering the conformation of bacteria or an interaction with neutrophils was not discerned, but an effect on bacteria seems likely given that SALSA's SRCR domains recognize and bind a wide range of microbial surface structures [2]. Different from a previous study that used the chain-forming Streptococcus gordonii, the current study used S. aureus, which naturally form aggregates [9]. ...
Full-text available
Salivary scavenger and agglutinin (SALSA) is a secreted protein with various immunomodu-latory roles. In humans, the protein agglutinates and inactivates microorganisms, and inhibits the release of pro-inflammatory cytokines. Saliva, which is rich in SALSA, accelerates bacterial phagocytosis, but SALSA's contribution is unclear. In horses, the functions of SALSA in inflammation remain undetermined, so they were investigated through phagocy-tosis and cytokine assays. Equine SALSA was purified from duodenal tissue, which contains abundant SALSA. To assess phagocytosis, fluorescently-labelled bacteria were incubated with 20, 10, 5, or 2.5 μg/mL of SALSA or phosphate buffered saline (PBS), and then incu-bated at 37˚C or on ice with whole blood from seven healthy horses. Fluorescence was measured by gating on neutrophils using a flow cytometer, and compared between groups. To assess effects on cytokine production, alveolar macrophages were isolated from bronch-oalveolar lavage fluid of five healthy horses and cultured in serum-free media for 24 hours with different concentrations of SALSA plus 1 μg/mL lipopolysaccharide (LPS), only LPS, or only media. Cytokines were measured in supernatant using an equine-specific multiplex bead immunoassay. There was significantly greater phagocytosis in samples incubated at 37˚C compared to incubation on ice. Samples incubated with 20 μg/mL of SALSA at 37˚C had less phagocytosis compared to samples with 10 or 2.5 μg/mL SALSA, or PBS. Alveolar macrophages incubated with SALSA plus LPS released significantly less CXC motif chemo-kine ligand 1, interleukin-8, interleukin-10, and tumor necrosis factor α, and more granulo-cyte colony stimulating factor (G-CSF), compared to macrophages incubated with LPS alone. These findings indicate anti-inflammatory effects, which may be due to interference with toll-like receptor 4 recognition of LPS or downstream signaling. Increase in G-CSF following incubation with SALSA suggests a novel mechanism for immunoregulation of alveo-lar macrophages by SALSA, addressing a knowledge gap regarding its functions in horses.
Full-text available
Lysyl Oxidase Like 2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises five lysine tyrosylquinone (LTQ)-dependent copper amine oxidases in humans. In 2003, LOXL2 was first identified as a promoter of tumour progression and, over the course of two decades, numerous studies have firmly established its involvement in multiple cancers. Extensive research with large cohorts of human tumour samples has demonstrated that dysregulated LOXL2 expression is strongly associated with poor prognosis in patients. Moreover, investigations have revealed the association of LOXL2 with various targets affecting diverse aspects of tumour progression. Additionally, the discovery of a complex network of signalling factors acting at the transcriptional, post-transcriptional, and post-translational levels has provided insights into the mechanisms underlying the aberrant expression of LOXL2 in tumours. Furthermore, the development of genetically modified mouse models with silenced or overexpressed LOXL2 has enabled in-depth exploration of its in vivo role in various cancer models. Given the significant role of LOXL2 in numerous cancers, extensive efforts are underway to identify specific inhibitors that could potentially improve patient prognosis. In this review, we aim to provide a comprehensive overview of two decades of research on the role of LOXL2 in cancer.
Streptococcus gordonii is a member of the viridans streptococci and is an early colonizer of the tooth surface. Adherence to the tooth surface is enabled by proteins present on the S. gordonii cell surface, among which SspB belongs to one of the most well studied cell-wall-anchored adhesin families: the antigen I/II (AgI/II) family. The C-terminal region of SspB consists of three tandemly connected individual domains that display the DEv-IgG fold. These C-terminal domains contain a conserved Ca ²⁺ -binding site and isopeptide bonds, and they adhere to glycoprotein 340 (Gp340; also known as salivary agglutinin, SAG). Here, the structural and functional characterization of the C 123 SspB domain at 2.7 Å resolution is reported. Although the individual C-terminal domains of Streptococcus mutans AgI/II and S. gordonii SspB show a high degree of both sequence and structural homology, superposition of these structures highlights substantial differences in their electrostatic surface plots, and this can be attributed to the relative orientation of the individual domains (C 1 , C 2 and C 3 ) with respect to each other and could reflect their specificity in binding to extracellular matrix molecules. Studies further confirmed that affinity for Gp340 or its scavenger receptor cysteine-rich (SRCR) domains requires two of the three domains of C 123 SspB , namely C 12 or C 23 , which is different from AgI/II. Using protein–protein docking studies, models for this observed functional difference between C 123 SspB and C 123 AgI/II in their binding to SRCR 1 are presented.
Cell membrane-bound serine proteases are important in the maintenance of physiological homeostasis. Hepsin is a type II transmembrane serine protease highly expressed in the liver. Recent studies indicate that hepsin activates prohepatocyte growth factor in the liver to enhance Met signaling, thereby regulating glucose, lipid, and protein metabolism. In addition, hepsin functions in nonhepatic tissues, including the adipose tissue, kidney, and inner ear, to regulate adipocyte differentiation, urinary protein processing, and auditory function, respectively. In mouse models, hepsin deficiency lowers blood glucose, lipid, and protein levels, impairs uromodulin assembly in renal epithelial cells, and causes hearing loss. Elevated hepsin expression has also been found in many cancers. As a type II transmembrane protease, cell surface expression and zymogen activation are essential for hepsin activity. In this review, we discuss the current knowledge regarding hepsin biosynthesis, activation, and functions in pathobiology.
The first months of life represent a crucial time period for an infant. Alongside establishing the early microbiome, the mucosal immunological homeostasis is being developed. Both processes may be perturbed in prematurely born infants. The glycoprotein SALSA plays a role in mucosal inflammation and microbial clearance. It is one of the most abundant molecules on the intestinal mucosal surfaces in early life. SALSA binds to many types of microbes and host defence molecules like IgA, C1q and collectin molecules. We here describe the development in faecal SALSA levels during the first three months of life. During these 90 days, the median SALSA level in full‐term babies decreased from 1100 μg/mL (range 49‐17 000 μg/mL) to 450 μg/mL (range 33‐1000 μg/mL). Lower levels of SALSA were observed in prematurely born infants in the same time period. Our novel observation thus indicates an impact of prematurity on an important component of the infant intestinal immune system. Changes in SALSA in early life may have an effect on the early establishment of the human microbiome.
Full-text available
Since its discovery, over 30 years ago, CD5 has been used as a marker to identify T cells, B1‐a cells, and B cell chronic lymphocytic leukemia cells. Throughout the years, many studies have described the functional relevance of CD5 as a modulator of T and B cell receptor signaling. However, it has not been until recent years that CD5 has emerged as a functional receptor in other areas of the immune system. Here, we review some of the most important aspects of CD5 as a modulator of TCR and BCR signaling, cell survival receptor both in T and B cells during health and disease, as well as the newly discovered roles of this receptor in thymocyte selection, T cell effector differentiation, and immune tolerance. CD5 was found to promote T cell survival by protecting autoreactive T cell from activation‐induced cell death, to promote de novo induction of regulatory T cells in the periphery, to modulate Th17 and Th2 differentiation, and to modulate immune responses by modulating dendritic cell functions. CD5 is overexpressed in Tregs and Bregs, which are fundamental to maintain immune homeostasis. The newly established roles of CD5 in modulating different aspects of immune responses identify this receptor as an immune checkpoint modulator, and therefore it could be used as a target for immune intervention in different pathologies such as cancer, autoimmune diseases or infections. Review on how CD5 regulates the balance between immunity and tolerance by modulating T/B cell signaling, T/B cell survival and T effector/Treg differentiation
Full-text available
The degree of evolutionary conservation of an amino acid in a protein or a nucleic acid in DNA/RNA reflects a balance between its natural tendency to mutate and the overall need to retain the structural integrity and function of the macromolecule. The ConSurf web server (, established over 15 years ago, analyses the evolutionary pattern of the amino/nucleic acids of the macromolecule to reveal regions that are important for structure and/or function. Starting from a query sequence or structure, the server automatically collects homologues, infers their multiple sequence alignment and reconstructs a phylogenetic tree that reflects their evolutionary relations. These data are then used, within a probabilistic framework, to estimate the evolutionary rates of each sequence position. Here we introduce several new features into ConSurf, including automatic selection of the best evolutionary model used to infer the rates, the ability to homology-model query proteins, prediction of the secondary structure of query RNA molecules from sequence, the ability to view the biological assembly of a query (in addition to the single chain), mapping of the conservation grades onto 2D RNA models and an advanced view of the phylogenetic tree that enables interactively rerunning ConSurf with the taxa of a sub-tree.
Full-text available
The Dali server ( is a network service for comparing protein structures in 3D. In favourable cases, comparing 3D structures may reveal biologically interesting similarities that are not detectable by comparing sequences. The Dali server has been running in various places for over 20 years and is used routinely by crystallographers on newly solved structures. The latest update of the server provides enhanced analytics for the study of sequence and structure conservation. The server performs three types of structure comparisons: (i) Protein Data Bank (PDB) search compares one query structure against those in the PDB and returns a list of similar structures; (ii) pairwise comparison compares one query structure against a list of structures specified by the user; and (iii) all against all structure comparison returns a structural similarity matrix, a dendrogram and a multidimensional scaling projection of a set of structures specified by the user. Structural superimpositions are visualized using the Java-free WebGL viewer PV. The structural alignment view is enhanced by sequence similarity searches against Uniprot. The combined structure-sequence alignment information is compressed to a stack of aligned sequence logos. In the stack, each structure is structurally aligned to the query protein and represented by a sequence logo.
Full-text available
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 available
CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Full-text available
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 exceptionally 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 secretory 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.
Full-text available
The protein deleted in malignant brain tumors (DMBT1) and the trefoil factor (TFF) proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human DMBT1(gp340). Analysis of lung washings and colon tissue extracts by Western blotting in the unreduced state, two antibodies (Hyb213-1 and Hyb213-6) reacted with a double band of 290 kDa in lung lavage. Hyb213-6, in addition, reacted against a double band of 270 kDa in colon extract while Hyb213-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340) positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue sections from patients with ulcerative colitis (p<0.0001) and Crohn's disease (p = 0.006) compared to normal colon tissue. Immunohistochemical analysis of trefoil factor TFF1, 2 and 3 showed that TFF1 and 3 localized to goblet cells in both normal colon tissue and in tissue from patients with ulcerative colitis or Crohn's disease. No staining for TFF2 was seen in goblet cells in normal colon tissue whereas the majority of tissue sections in ulcerative colitis and Crohn's disease showed sparse and scattered TFF2 positive goblet cells. DMBT1 and TFF proteins did therefore not co-localize in the same cells but localized in adjacent cells in the colon. The interaction between DMBT1(gp340) and trefoil TFFs proteins was investigated using an ELISA assay. DMBT1(gp340) bound to solid-phase bound recombinant dimeric TFF3 in a calcium dependent manner (p<0.0001) but did not bind to recombinant forms of monomeric TFF3, TFF2 or glycosylated TFF2. This implies a role for DMBT1 and TFF3 together in inflammatory bowel disease.
Neurotrypsin (NT) is a multi‐domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C‐terminal serine‐protease (SP) domain. However the purpose of NT's remaining 3‐4 scavenger receptor cysteine‐rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT‐SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca²⁺ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners. This article is protected by copyright. All rights reserved.
It is becoming increasingly clear that the connections between our immune system and the microbiota colonizing us have a tremendous impact on human health. A number of innate molecular defence mechanisms cooperate to selectively target unwanted microorganisms at the mucosal surfaces. Amongst others these include the complement system, IgA and the SALSA molecule. The salivary scavenger and agglutinin (SALSA), also known as deleted in malignant brain tumors 1 (DMBT1), salivary agglutinin (SAG) or gp340 is a multifunctional molecule with important functions in innate immunity, inflammation and epithelial homeostasis. The SALSA protein is expressed at most mucosal surfaces, where it is one of the most abundant proteins. In the fetal meconium and infant intestine it may constitute even up to 10% of the total protein amount. SALSA is found either directly associated with the epithelial surface or secreted into the lining fluids. In the fluid-phase SALSA interacts with a number of bacterial and viral organisms, as well as with endogenous ligands, including IgA, lactoferrin, surfactant proteins and complement components. While complement has been shown to impact the mucosal environment, this remains an area of limited research. The multiple interactions of the SALSA molecule provide a scaffold, where this potent defence system may engage in cooperative microbial clearance together with corresponding mucosal host ligands.
CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100-kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6.