Polish Journal of Veterinary Sciences Vol. 9, No.

4 (2006), 265-275

Review article

Conglutinin, CL-43 and CL-46 – three bovine

collectins

M. Dec, A. Wernicki

Agricultural Academy, Faculty of Veterinary Medicine, Institute of Infective and Invasive Diseases,
Department of Veterinary Prevention
Akademicka 12, 20-033 Lublin, Poland

Abstract

Conglutinin, collectin-43 (CL-43) and collectin-46 (CL-46) are serum proteins characteristic for
Bovidae. They belong to collectins – family of oligomeric proteins composed of trimeric subunits
containing collagen-like sequences joined to C-type lectin domains. The genes encoding conglutinin,
CL-43 and CL-46 are located on the bovine chromosome 28, and phylogenetic analysis indicates their
common origin – from the lung surfactant protein D gene. Northern blot or immunocytochemical
analysis confirm biosynthesis of bovine collectins mainly in the liver (conglutinin, CL-43) and in the
thymus (CL-46).
The level of conglutinin in the serum of dairy cows depends on many factors such as breeding, the
season of the year, the stage of the reproductive cycle and infection.
The collectins are involved in the innate immune defense. They bind to microbial surface carbo-
hydrates inducing aggregation and, thereby, impeding infectivity. On the other hand the destruction
of pathogens occurs due to stimulation of effector cells. CL-43 as well as conglutinin, binds to the
collectin receptor (C1qR) localized on many types of cells identified as a surface variant of cal-
reticulin. Conglutinin and CL-43 show antiviral activities towards influenza A virus and rotaviruses.
Conglutinin also displays protective activity against bacterial infections.

Key words: conglutinin, CL-43 and CL-46, structure and biological activity.

Introduction (surfactant protein A) and SP-D (surfactant protein

Collectins (collagen-like lectins) is a subgroup of
mammalian C-type (Ca+2- dependent) lectins in-
volved in innate defense mechanisms. They have simi-
lar structures characterized by the presence of col-
lagen-like sequences and globular carbohydrate rec-
ognition domains (CRDs). The collectins include the
serum proteins: mannan binding protein (MBP)
– called also mannan binding lectin (MBL), con-
glutinin, collectin of 43 kDa (CL-43), collectin of 46
kDa (CL-46) and two lung surfactant proteins: SP-A
D). Novel collectin liver 1 (CL-L1) (Ohtani et al.
1999) and collectin placenta 1 (CL-P1) (Ohtani et al.
2001) are found in the cytosol and cell membrane,
respectively. Collectins play a key role in the first line
of defense against invading microorganisms, as dem-
onstrated by the experiments with transgenic mice de-
ficient in MBP (Shi et al. 2004), SP-A (Atochina et al.
2004) or SP-D (Madan et al. 2005). Such animals,
likewise people with the mutation of gene for MBP,
show increased susceptibility to bacterial and viral in-
fections (Koch et al. 2001, Thiel et al. 2006). Collec-

Correspondence to: M. Dec, e-mail: marta.dec@ar.lublin.pl

266
tins display the affinity to surface microbial carbohy-
drates. They bind to a variety of Gram-positive and
Gram-negative microorganisms, yeasts and parasites,
enveloped viruses and trigger the killing and clearance
mechanisms through the activation of complement or
interaction with specific receptors on phagocytic cells.
Individual collectins, because of their sequence dif-
ferences in the carbohydrate recognition domains,
play particular roles in the host defense. Collectins
bind to carbohydrates on surface of various microor-
ganisms and to specific receptors on phagocytic cells,
thus accelerating microbial clearance (Lu 1997).
Conglutinin, CL-43 and CL-46 are serum proteins
characteristic for Bovidae. It can be connected with
the fact that these animals live in symbiosis with an
enormous number of microbes in their rumen. It is
supposed that serum collectins provide the first line of
defence against these microbes (van de Wetering et
al. 2004).
Conglutinin was found not only in the cattle but
also in a one-humped camel (Camelus dromaderius)
(Kakoma and Kinyanjui 1974) and in a number of
African ruminants – the African buffalo, water buck,
Uganda kob, Kenya kob, dik-dik, topi, and Jackson;s
hartebeest. In this last species conglutinin had a par-
ticularly high titre. It remains unclear why conglutinin
does not occur in the serum of nonruminant herbi-
vores like horses, which also live in symbiosis with
microorganisms living in their caecum. The mice’s,
rat’s, rabbity, guinea pig’s, equine, feline, canine, por-
cine, and human sera do not show any conglutinin
activity (Lachmann 1967).
Structure of bovine collectins
The collectins are large molecules with molecular
masses ranging from about 100 kDa to more than
1000 kDa (Hansen and Holmskov 1998). The basic
functional unit of collectins is a trimer of three ident-
ical monomers. Such structure is important to their
proper function. The carbohydrate affinity of a single
collectin CRD is probably weak but the trimeric ar-
rangement permits a stronger interaction between
collectin and carbohydrate-containing target microor-
ganism. Most of the collectins are built of several
trimer units, what enables them to cross-link several
target particles and perhaps also to interact simulta-
neously with host cells (Hakansson and Reid 2000).
The number of trimeric units per molecule differs
among the collectins. Conglutinin contain 4 trimeric
units, Cl-43 – 1 unit, and CL-46 may also form tet-
ramers (Hansen et al. 2002). In the monomeric
subunit, four structural domains can be distinguished:
– an N-terminal cysteine-rich domain,
– a collagen-like domain,
– α-helical neck domain, and
Dec M., Wernicki A.
– C-type lectin domain, also known as CRD (van de
Wetering et al. 2004).
The trimeric subunits are stabilized by non-
covalent interactions (strong hydrophobic forces in
α-helical bundle) and covalently by disulfide bridges
between cysteine residues in the N-terminal or col-
lagen regions (Fig. 1).
The CRD is responsible for selective binding of
collectin to carbohydrates in the presence of calcium
ions. The collagen domain is involved in receptor-me-
diated effects of collectins, and consists of repeated
motifs of Gly-X-Y, where X and Y can be any amino
acid. The collagen monomer chains are coiled around
each other forming a stable strained domain generally
organized into “bundle of tulips” or X-like structures
(Lu 1997).
The primary structure of conglutinin has been re-
solved first by complete sequencing of the protein
(Lee et al. 1991a), and subsequently through the se-
quencing of cDNA (Lu et al. 1993, Suzuki et al. 1993,
Liou et al. 1994).
The molecule of conglutinin contains 351 amino
acid residues including 55 apparent Gly-X-Y repeats
with two interruptions. This 171-residue-long col-
lagen-like domain separates a short noncollagen
NH2-terminal region of 25 residues from the 155-resi-
due-long globular COOH terminus. The structural
analysis of conglutinin showed that there are 7 cys-
teine residues present which are capable of forming
inter – and/or intramolecular disulfide bridges. The
differences between calculated – 39.7 kDa (Holmskow
2000) and observed – 750 kDa (Lachmann 1967) mol-
ecular weights are due to posttranslational modifica-
tion such as glycosylation (Lee et al. 1991a). The col-
lagen-like region of conglutinin contains eight hy-
droxylysine residues, which are possible glycosylation
sites. Deglycosylation and glycan differantiation
analysis of conglutinin revealed the presence of O-lin-
ked glycans of β(1-3)-GalNAc and α-(2-3) linked sia-
lic acid type. No N-linked glycans were detected (An-
dersen et al. 1992a).
In the electron microscope, conglutinin is ob-
served as an X-shaped, tetrameric molecule with four
globular heads, each connected to central “hub” by
four elongated collagen arms (Fig. 1). The average
overall diameter of the tetramer is 96 nm. It is poss-
ible that the conglutinin molecules adhere to each
other in the central lob region, forming rosette-like
aggregates (Andersen et al. 1992a).
Although the arms are flexible, distinct bends are
occasionally seen at two positions. One bend is
located a few nanometers from the central lobe and
the other approximately 25 nm from the “hub”. These
flexible regions are consistent with two interruptions
in collagen-like domain at position 38 (Cys replacing
Gly) and at residue 123 [Gly replacing any other
amino acid (X)].
Twins rate in the black-and-white...
Fig. 1. Molecular structure of collectins. A: Three identical polypeptide chains form stable tensile domain as a result of covalent
and hydrophobic interactions. B: Conglutinin consisting of the tetrameric structural units, is assembled into “X-like” shape.
CL-43 molecules exist as monomers. (Hoppe and Reid 1994).
In Superose 6 column conglutinin is separated
into tree fractions. Electron microscopy showed that
peak 1 contains mainly tetramers, peak 2 – mainly
dimers and peak 3 – only monomers, which are the
products of protein degradation in the collagen-like
region between Arg 54 and Ala 55. The amount of
such truncated fraction (40 kDa) in a sample of purifi-
ed conglutinin increases slowly during storage at 4oC.
The cause of such a specific degradation is not clear
but may involve the presence of a low level of serum
proteases, such as plasmin, in the conglutinin prepara-
tion (Lu et al. 1993).
CL-46 shows high resemblance with conglutinin.
The molecule contains 371 amino acid (aa) residues:
25 aa N-terminal segment, 57 Gly-X-Y repeats, 28 aa
neck region, and a CRD of 127 aa. Potential
N-glycosylation site is localized at asparagine 90 in the
collagen-like region (Hansen et al. 2002).
267
Collectin-43 is found only as a single subunit com-
posed of three identical polypeptide chains (Fig. 1).
Protein sequencing, cDNA cloning and reverse tran-
scriptase PCR techniques have shown that CL-43 con-
tains 301 amino acid residues, and cDNA is entirely
consistent with the protein sequencing. The difference
in length between conglutinin and CL-43 is caused by
two deletions in the collagen region of CL-43, which
only contains 38 Gly-X-Y triplets compared with 57
Gly-X-Y triplets of conglutinin. The amino acid se-
quence of CL-43 showed 74% identity with con-
glutinin and 70% with bovine SP-D (Lim et al. 1994,
Holmskov et al. 1995). CL-43 like conglutinin does
not posses N-linked carbohydrate, but it contains hy-
droxylysine residues in the collagen-like region that
are potential O-glycosylation sites (Holmskov et al.
1995).
On SDS/PAGE under reducing conditions the
268
purified collectin-43 shows as double band of about 43
kDa, with the upper band representing the intact mol-
ecule and the lower band being a truncated form that
lacks nine the N-terminal nine amino acid residues.
Under non-reducing conditions, only one band of 120
kDa. In two-dimensional gel electrophoresis CL-43 is
seen as two isoforms, with pI (isoelectric point) values
of 4.9 and 5.3, corresponding to the native and the
truncated form of the molecule, respectively (Hol-
mskov et al. 1993, Holmskov et al. 1995).
Reduced conglutinin shows as a major band of 44
kDa (native form) and minor band of 40 kDa (trun-
cated form). Under nonreducing conditions con-
glutinin is seen as a ladder (Krogh-Meibom et al.
2004a) pattern with bands ranging from 32 kDa (Lu et
al. 1993) to 300 kDa (Andersen et al. 1992b).
Sites of conglutinin, CL-43 and CL-46
biosynthesis
The genes encoding conglutinin, CL-43 and CL-46
are located on the bovine chromosome 28 at position
q1.8 (Gallagher et al. 1993, Liou et al. 1994, Hansen
et al. 2002, Hansen et al. 2003 ). The phylogenetic
characterization of these three bovine collectins genes
indicates that they are structural descendants of an-
other collectin – lung surfactant protein D (SP-D) as
a consequence of complete or partial ancestral SP-D
gene duplication after the divergence of Bovidae from
other mammals (Holmskov 2000, Hansen et al. 2003,
Gjerstorff et al. 2004). During the studies on the bov-
ine DNA encoding collectins some authors identified
a gene that potentially encodes a novel con-
glutinin-like collectin (COLEC14) (Gjerstorff et al.
2004).
Immunocytochemical analysis showed the pres-
ence of conglutinin in many tissues, but the intensity
of cytoplasmatic staining in all hepatocytes suggests
the liver as the principal site of synthesis of this lectin.
Conglutinin is also associated with follicular dendritic
cells in the germinal centres of the spleen, tonsils and
lymph nodes, and with macrophages from the liver,
lung, thymus and spleen. Conglutinin staining has also
been present in the glomerulus of the kidney and in
endothelial cells. Moreover, this collectin internalized
in the course of phagocythosis is detected by im-
munostaining in macrophage’s phagolisosomes (Hol-
mskov et al. 1992). Northen blot analysis revealed the
presence of mRNA for conglutinin only in the bovine
liver, a weak signal was present also in the sheep liver
(Lu et al. 1993).
Northern-blot analysis on total RNA, purified
from various tissues from cattle, sheep, humans, rats
and mice, showed a strong signal of approx. 1.8 kb in
bovine liver RNA (Lim et al. 1994). A weak signal of
similar size was also observed in the sheep liver and in
Dec M., Wernicki A.
the lung RNAs of all the species tested (Lu et al.
1993). Similar observations were reported by Lim et
al. (1993), but, unexpectedly, the strongest signal was
obtained not from the liver, but from bovine lung
RNA.
CL-43 is synthesized only in the bovine liver, and
contrary to conglutinin, no signal was detectable in
mRNA from sheep, human, rat or mouse tissues. This
suggests that CL-43 appeared later in evolution than
conglutinin (Lim et al. 1994).
Hansen et al. (2002) showed that the dominant
place of CL-46 expression is the thymus. They also
observed the production of this collectin in the liver
and moderate expression was detected in the mam-
mary gland and in tissues throughout the digestive
system.
Serum level of bovine collectins
The level of conglutinin in the serum of dairy cows
varies with the season of the year, stage of the repro-
ductive cycle and is altered by infectious agents. In-
gram and Barnum’s (1965) studies, using mainly Hol-
stein cows, demonstrated the titer of conglutinin in
the sera of adult cattle in the range from 0 to 5120.
These data were estimated by the agglutination of
alexinated erythrocytes – a method described by
Coombs et al. (1961). Recently, there has been con-
structed a more efficient time-resolved immunof-
luorometric assay (TRIFMA) for quantification of
conglutinin or CL-43 based on poly- and monoclonal
antibodies (Krogh-Meibom et al. 2004a).
The low point of conglutinating activity occurs in
late winter and spring, and the peak activity – in fall
and early winter. The differences in mean conglutinin
titers at these two periods are greater than fourfold.
The major increase occurs several weeks after the
cows are pastured – that indicates that good nutri-
tional conditions increase the concentration of con-
glutinin (Ingram et al. 1971a). The additional factors
inducing such titer fluctuations can be ambient tem-
perature or duration of daylight (Ingram and Barnum
1965). No significant changes in conglutinin or CL-43
level due to circadian rhythm were noticed
(Krogh-Meibom et al. 2004a).
Large alterations in conglutinin level are connec-
ted with pregnancy and abortion. A marked decrease
in conglutinatin activity occurs shortly before calving
(begining from 4 weeks before term of calving) and
lasts until 2 weeks after parturition. From this low
point the serum level increases quite rapidly and
reaches normal (high) level at about 21 weeks after
parturition (Ingram and Mitchell 1970). Similar
changes are associated with abortion – marked drop
in conglutinin serum level was observed in the week of
miscarriage, and between 2 and 4 weeks after collectin
Twins rate in the black-and-white...
was not detectable at all (Ingram and Mitchell 1971b).
Such a suppression in conglutinin concentrations can
probably be associated with changes in gonadal hor-
mones or corticosteroids level, which occur due to
gestation.
Most of newborn calves do not have conglutinin
in their serum prior to the feeding of colostrum.
After feeding colostrum, the conglutinin appears in
the serum of young animals and increases until 80
weeks of age when adult levels are approached. The
conglutinating activity in the milk whey (obtained
from colostrum and milk) was not correlated with
the conglutinin serum level, season of the year, stage
of lactation or gestation (Ingram 1965, Ingram 1970).
The reasons for alterations in conglutinating ac-
tivity in bovine serum are diseases as pneumonia,
metritis or other nondiagnosed acute infections.
A significant decrease in conglutinin titer appears
1 week before the disease is recognized and it is held
down for 1-2 weeks after the first recognition. Some
authors suggest that a reason for these changes could
be marked hyperthermia, which arised in all affected
cows. However, the most probable cause of the low
level of conglutinin during acute infections is its ab-
sorption from the serum by activated complement
components iC3b or directly by microorganisms, al-
though down-regulation of gene is also possible. Mi-
nor or local infections, such as mild forms of mastitis
or foot rot (necrotic pododermatitis, foul foot) do
not have a significant effect on the level of serum
conglutinin. No changes occurred in the case of lym-
phosarcoma either (Ingram and Mitchell 1971b).
A fall in conglutinin titer was demonstrated during
experimental infections with Anaplasma marginale
and Cowdria ruminantium (intracellular rickettsial
parasites) (Rose et al. 1978, du Plessis 1985).
Reports published by Holmskov et al. (1998)
have shown that the level of conglutinin in the cattle
is determined genetically. The geometric mean con-
centration in males (15.5 µg/ml) is significantly high-
er than in females (8.1 µg/ml), and about 2% of ani-
mals are probably homozygous for a single recessive
allele causing low concentrations of conglutinin. The
level of conglutinin differs in calves from various
farms probably because of different breeding and
management practices (Purdy et al. 2000).
Moreover, the low conglutinin level is associated
with an increased susceptibility to respiratory infec-
tion. These findings suggest that the serum con-
glutinin concentration may be helpful in the selec-
tion against resistance to infectious disease (Hol-
mskov et al. 1998). On the other hand Purdy et al.
(2000) did not detect any significant differences in
conglutinin titers among transported calves asso-
ciated with the bovine respiratory disease complex
morbidity, vaccination status against Mannheimia
haemolytica or sex.
269
Collectin receptors
The selective affinity of collectin to carbohydrates
suggests reactivity of the lectin domains with a large
range of microrganisms. There are many indications
that they act as opsonins and mediate biological func-
tions by futher interactions with phagocytic cells.
Several types of receptors for collectins are found
associated with the membranes of different cells (van
de Wetering et al. 2004). They are responsible for
stimulating phagocytes and promoting killing and
clearance of microorganisms.
Due to the structural similarity between C1q and
the collectins the receptor for C1q – C1q-R was
identified as general collectin receptor. Some authors
even classify C1q to the collectin family (Favoreel et
al. 2003). Its molecule, resembling a bunch of tulips, is
organized into six subunits containing triple-helical
stalks throughout their collagen-like domains
(Gly-X-Y) and globular heads in the C-terminal re-
gions. Each subunit is composed of three chains – A,
B and C. However, C1q lacks a lectin domain, and
contrary to collectins, it binds to protein motifs in IgG
or IgM rather than to carbohydrates (Kishore et al.
2004). Purified C1q-R, apart from C1q, binds also
conglutinin, CL-43 as well as MBL and SP-A (Mal-
hotra et al. 1990, Malhotra et al. 1992) via their col-
lagenous regions, where charges of amino acid resi-
dues are clustered (Malhotra et al. 1992, Malhotra et
al. 1993).
In the case of conglutinin such region of charge
density occurs close to the N-terminus of protein. The
studies on the form of truncated conglutunin, which
has 55 amino acids missing from the N-terminal do-
main, strongly indicate that receptor-ligand interac-
tion is mediated via N-terminal region of conglutinin.
The absence of this sequence also prevents the forma-
tion of four-subunit structure found in normal con-
glutinin (Malhotra et al. 1993).
The collectin receptor was purified from
B cell-derived lymphoblastoid Raji cell line (Gheb-
rehiwet et al. 1984). C1q-R shares a high degree of
amino acid homology with calreticulin (CRT) (Mal-
hotra at al. 1993) – the protein acts as a chaperonin
for nascent protein in endoplasmic reticulum
(Michalak et al. 1999). Due to the identical N-ter-
minal sequences and the same mobility on
SDS-PAGE (56.000 Da under reducing conditions)
(Malhotra at al. 1993) it is belived that collectin recep-
tor is a surface variant/isoform of calreticulin. Fur-
thermore, both the human C1q-R and CRT bind C1q,
collectins (MBL, SP-A, CL-43, but not SP-D) and col-
lagens with identical characteristics, and the binding
site has been localised within putative C1r/C1s mod-
ule (CUB domain) of calreticulin (Eggleton et al.
1994a, Stuart et al. 1996, Stuart et al., 1997).
270
The collectin receptor is found in a wide variety of
cell types, including human tonsil lymphocytes, the
monocytic cell line U937 (Malhotra and Sim 1989),
endothelial cells and platelets (Peerscheke et al. 1993)
as well as the lung alveolar type-II cell line A549
(Malhotra et al. 1992). Flow cytometric analysis detec-
ted calreticulin on the surface of neutrophil, which
decreased during stimulation probably as a conse-
quence of shedding – calreticulin is present in the cell
supernatants of stimulated cells (Eggleton et al.
1994a, Eggleton et al. 1994b, Kishore et al. 1997).
Since calreticulin has neither a transmembrane
domain nor glycosylphosphatidylinisotol (GPI) an-
chor attachment site it must utilize an adaptor mol-
ecule to attach to the plasma membrane. Ghiran et al.
(2003) has shown that CRT is associated with CD59
on neutrophil surface. Calreticulin is also functionally
associated with macrophage receptor CD91. The liga-
tion of CRT to C1q or collectin-opsonized apoptotic
cells induces phagocytosis of the apoptotic cells by
macrophages through CD91-dependent mechanism,
where CD91 is thought to be involved in the transduc-
tion of the signal initiating engulfment (Ogden et al.
2001, Vandivier et al. 2002).
C1q and collectins, namely MBL and SP-A, en-
hance the phagocythosis of sheep erythrocytes op-
sonized with antibody or C4b complement compo-
nent. This enhancement is caused via direct interac-
tions between collectins and 126 000 Mr phagocyte
surface glicoprotein – C1qRp, and is not due to inter-
action between the surface-bound C1q and the
EA-IgG (erythrocyte-associated IgG). (Bobak et al.
1987, Tenner et al. 1989, Tenner et al. 1995). Such
receptor is localized on human cells of myeloid lin-
eage (monocytes or neutrophils), the U937, THP-1,
and K562 cell lines and platelets (Nepomuceno and
Tenner 1998). Experiments conducted with purified
fragments of C1q and MBL suggest that the phago-
cytosis enhancement signal resides in the collagen-like
tail domain of the molecules (Bobak et al. 1987, Guan
et al. 1994, Arora et al. 2001), so it is very probable
that bovine collectins also show affinity to C1qRp.
Carbohydrate selectivity
An important property of the collectins is their
capability of binding carbohydrates. Natural
(poly)saccharide ligands for the collectins are nor-
mally attached to the surface of microorganisms. Stu-
dies demonstrated that high-affinity interactions be-
tween microorganisms and collectins depend on the
density of carbohydrate ligands on the microbial sur-
face (Lee et al. 1991b), and on the degree of
oligomerization of the collectin (Hartshorn et al.
1996, Hartshorn et al. 1998). Clustering of glycop-
Dec M., Wernicki A.
roteins or glycolipids on the surface of microorgan-
isms allows for the simultaneous binding of multiple
CRDs of one assembled collectin molecule (van de
Wetering et al. 2004).
Differences in the glycosylation pattern of invad-
ing microorganisms and the host cells allow to distin-
guish between the alien and own carbohydrate struc-
tures. Reaction of saccharides binding to collectins is
Ca+2-dependent and EDTA-inhibitable (Reading et
al. 1998).
Conglutinin, collectin-43 and CL-46 bind to
a range of sugar residues including mannose, fucose,
glucose, maltose, GlcNAc or ManNAc. The degree of
affinity of particular collectins to different sugars is
shown in Tab. 1.
Carbohydrates bound by collectins has been struc-
turally characterized in several complexes with the use
of mammalian or chicken MBP. These studies indi-
cate that binding of carbohydrates involves hydrogen
bonds and van der Waal’s interactions, and is stabil-
ized by coordination bonds to the calcium ions. The 3-
and 4-OH groups of the sugar directly coordinate
Ca+2 and form hydrogen bonds with amino acids that
also serve as Ca+2 ligands (Lee et al. 1991, Weis et al.
1992, Kolatkar and Weis 1996, Ng et al. 1996, Hakan-
sson and Reid 2000).
Amino acid analysis and monosaccharide inhibi-
tion studies indicate that all collectins have man-
nose-type CRDs with the exception of collectin pla-
centa 1, such structural analysis indicates the prefer-
ence of galactose over mannose althought this has not
been tested yet (Ohtani et al. 2001, van de Wetering
2004).
Conglutinin exhibits a unique property of aggluti-
nation of erythrocytes preincubated with antibodies
and complement, which is not shared by any of the
other collectins. This reaction is dependent on the
presence of conglutinogen-activating factor in the
serum, now known as factor I. It cleaves the comple-
ment component C3b generating inactivated factor
iC3b, which subsequently opsonizes erythrocytes.
Conglutinin is able to binds such alexinated eryth-
rocytes. This calcium-dependent reaction is based on
selective reactivity between conglutinin and high man-
nose N-linked oligosaccharide on the α-chain of iC3b
at Asparagine[917], and can be inhibited by monosac-
charides, with N-acetyl-D-glucosamine being the most
potent (Hirani et al. 1985, Hirani et al. 1986, Laursen
et al. 1994). The affinity of conglutinin to oligosac-
charide on α-chain of iC3b is qualified by the pres-
ence of terminal α 1-2 linkages. A preference for the
terminal Man1-2 sequence was also observed during
the examination of the conglutinin binding to
neo-glycolipids (Mizuochi et al. 1989). However, con-
glutinin-N (lacking 54 N-terminal amino acids) had
neither conglutination activity (Lu et al. 1993) nor
binding activity to the sensitized erythrocyte-iC3b
Twins rate in the black-and-white...
Table 1. Comparison of bovine collectins carbohydrate selectivitya
Conglutinin GlcNac >>> ManN, L-Fuc, Man > Glc >> Mal, GlcN > GalN > Gal > Fuc >>>> GalNAc
rCL-46 GlcNac >>> ManNAc, Man > αMeMan > Mal, GlcN > Man, Glc, L-Fuc >> Gal > αMeGlu > Fuc
CL-43 Man >>> ManNAc >>> L-Fuc > GlcNAc >> Glc > Mal >>> Gal, Lac >>>> GalNAc
a
Carbohydrate specificities based on 50% of collectin binding (Haurum et al. 1993, Holmskov et al. 1993, Loveless et al. 1995,
Holmskov 2000, Hansen et al. 2002). >, >>, and >>> denote 10%, 50% and 100% higher than I50 value in comparison to the
previous saccharide.
complex, although it retained the binding activity to
mannan in solution and the original binding specificity
for N-acetylglucosamine (Kawasaki et al. 1993). An-
other study has revealed that conglutinin lacking the
N-terminal and collagenous domains does not com-
pletely lose conglutinating activity (Eda et al. 1996).
Thus, it is still unclear whether the overall molecular
structure of conglutinin is necessary for iC3b binding.
The conglutinin property to agglutinate erythrocytes
is used to purify and quantify (conglutinin binding as-
say) circulating immune complexes in patients with
different diseases (D’Amelio 1981, Araga et al. 1984,
Holmskov et al. 1992, Heinonen et al. 1993).
Moreover, conglutinin reacts with zymosan
– a cell-wall preparation from Saccharomyces
cerevisiae, and to mannan – solube yeast glycop-
roteins. The binding to zymosan is inhibited by
oligosaccharides with non-reducing terminal α 1-2 but
not α 1-3 mannosyl moieties. The conglutinin ability
to bind zymosan or iC3b is used to isolate this collec-
tin (Lachmann 1962, Andersen et al. 1992b).
Antiviral activity
The bovine collectins bind to the surface glycop-
roteins of several enveloped viruses, including influ-
enza virus, human immunodeficiency virus or herpes
simplex virus, as well as to the nonenveloped
rotavirus.
Conglutinin inhibits hemagglutination activity and
infectivity of influenza A virus (IAV) H1, H2 and H3
subtypes (Hartley et al. 1992, Wakamiya et al. 1992,
Hartshorn 1993). By using virus strains with specific
variations in glycosylation of the hemagglutinin mol-
ecule it has been shown that such inhibition is me-
diated by binding of conglutinin to high mannose
carbohydrate attachments on the viral hemagglutinin.
Through the same mechanism conglutinin caused the
aggregation of IAV particles (Hartshorn et al. 1993),
and because of that enhanced neutrophil respiratory
burst response to IAV or internalization of IAV (Har-
tshorn et al. 1997). IAV-induced H2O2 production is
mediated by cross-linking of sialylated neutrophil sur-
face receptors. Larger viral aggregations formed by
conglutinin, appear to cause increased H2O2 produc-
271
tion by provoking more extensive cross-linking of sia-
lylated receptors (Hartshorn et al. 1996). This theory
conforms with the studies on native and recombinant
CL-34. This lectin also shows antiinfluenza activity,
but contrary to conglutinin it does not induce viral
aggregation, and simultaneously is unable to enhance
IAV-induced neutrophil H2O2 production (Hartshorn
et al. 2002).
The findings of Eda et al. (1996) using recom-
binant conglutinin lacking the N-terminal and col-
lagen domain have shown that such incomplete mol-
ecule retains the ability to inhibit IAV hemagglutina-
tion activity. Moreover, this reaction can be blocked
by N-acetylglucosamine, and is dependent on divalent
cations (Wakamiya et al. 1992, Hartshorn et al. 1993).
These findings as well as research by Harshorn et al.
(2000) using chimeric protein made by fusing N-ter-
minus and collagen domain of surfactant protein
D and CRD of conglutinin confirm that the potent
IAV-neutralizing activity of conglutinin resides in its
neck region and carbohydrate recognition domain.
CL-43 and conglutinin have in vitro antiviral activ-
ity against rotaviruses which cause acute viral gastro-
enteritis (Ramig 2004). Both collectins show hema-
gglutination inhibition as well as neutralizing activity.
These properties are associated with binding of con-
glutinin and CL-43 through their lectin domains to
VP7 glycoprotein of rotavirus. Implicated in virulence
surface VP7 carries high-mannose type carbohydrate,
and the susceptibility of rotaviruses to collectin is de-
pendent on the number of glycosylation sites on VP7
molecule. CL-43 displays higher antiviral activity
against rotaviruses than conglutinin, and in the case of
influenza virus A/HKx31 the relation is inverse
(Reading et al. 1998).
Binding of conglutinin to herpes simplex virus
type 2 is Ca+2-dependent and inhibited by carbohy-
drates. However, when collectin was administered to
mice before virus inoculation, enhancement rather
than inhibition of virus replication was observed, in-
dicating that conglutinin may provide the virions with
an alternative port of entry into cells (Fischer et al.
1994).
The highly glycosylated envelope glycoprotein gp
160 of human immunodeficiency virus (HIV) interacts
with the CD4 molecule present on the surface of
272
CD4+ cells and is involved in the pathology of HIV
infection. Conglutinin peculiarly binds to recombinant
protein gp160. This reaction, as in the case of other
viruses, is calcium-dependent, is inhibited with
N-acetyl-D-glucosamine, and deglycosylation of
gp160 abrogates this reaction. These results indicate
that conglutinin may inhibit the HIV infection via ex-
clusion of interaction between viruses and CD4+ cells
(Andersen et al. 1991).
Antibacterial activity
Conglutinin was the first collectin shown to have
a protective effect against infection in vivo. In 1959
Ingram showed interrelations between the con-
glutinating activity in the animal serum and experi-
mental bacterial infection. The challenge of mice with
Salmonella typhimurium results in rapid reduction of
the level of serum conglutinin (Ingram 1959a). More-
over, prior injection of conglutinin causes higher sur-
vival rates in mice infected with virulent strains of
Salmonella typhimurium, Pasteurella septica, Klebsiella
pneumoniae, Listeria monocytogenes, Streptococcus
pneumoniae and Streptococcus pyogenes. The con-
glutinin preparations fail to protect mice against
a non-pathogenic species (E. coli) or avirulent strains
of pathogenic organisms (Ingram 1959b).
This evidence was later confirmed in vivo and in
vitro by Fris-Christiansen et al. (1990). Mice injected
subcutaneously with conglutinin before in-
traperitoneal injection of a highly virulent strain of S.
typhimurim displayed prolongation of survival. More-
over, conglutinin-treated mice showed a significantly
reduced number of bacteria in the blood as compared
to the control animals treated with saline.
In vitro experiments demonstrate that the antibac-
terial activity of conglutinin against Salmonella ty-
phimurium and E. coli requires the presence of both
an intact complement system and macrophages.
Serum deficient in factor I, which is necessary for gen-
eration of iC3b from C3b, could not support this reac-
tion. Also the presence of EDTA (chelator of divalent
ions) during the incubation of serum-pretreated bac-
teria with conglutinin abolished the conglutinin effect.
These results suggest that conglutinin binds to iC3b
deposited on the bacterial surface, and acts as a ligand
mediating contact between bacteria and effector cells.
It is very probable that conglutinin induces antibac-
terial activity by stimulating special receptors localiz-
ed on the surface of effector cells (Fris-Christiansen
et al. 1990).
The generation of oxygen metabolites by effector
cells is probably one of the mechanisms of antibac-
terial activity of conglutinin. The bovine conglutinin
enhances the respiratory burst of spleen cells stimu-
lated with E. coli in the presence of iC3b. A con-
Dec M., Wernicki A.
glutinin alone – without bacteria and normal serum
– does not evoke such reaction. Hence, it is speculated
that binding of this collectin to iC3b deposited on the
bacteria may allow the collagen domain of conglutinin
to adhere to the collectin receptor on the spleen effec-
tor cells (Friis et al 1991).
There are some unpublished relations concerning
antibacterial activities of recombinant CL-43. Collec-
tin-43 increases neutrophil uptake of E. coli, like IAV
particles, but without causing bacterial aggregation
(Hartshorn et al. 2002).
Cl-43 also shows antimicrobial properties towards
noncapsulated form of Cryptococcus neoformans. This
pathogenic yeast is commonly found in that form in
the environment and causes opportunistic infection.
CL-43 binds in a calcium-dependent manner to non-
capsulated C. neoformans provoking yeast agglutinat-
ion. Such reaction is abolished by selected saccharides
(Schelenz et al. 1995).
Conclusion
The serum collectins – conglutinin and CL-43,
play significant role in innate antimicrobial immune
defence system in Bovidae. They can aggregate patho-
gens, neutralize viruses, activate phagocytes and pro-
mote phagocytosis. Unfortunately, available data are
insufficient, particularly concerning CL-46, and fur-
ther studies are needed to fully understand the bio-
logical function of these proteins and assign the thera-
peutic implication.
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