(Received for publication, May 3, 1995; and in revised form, September 29, 1995 )
From the
Collectins are C-type animal lectins with both collagenous and
carbohydrate recognition domains and are involved in the first line
host defense against pathogens. We report here a novel
Ca-dependent and GlcNAc-binding lectin consisting of
subunits of 35 kDa (P35) with a collagen-like sequence. When P35 is
isolated from human serum, it forms a homopolymer by means of
intermolecular disulfide bonding, as is the case with collectins. P35
cDNA was cloned from a human liver cDNA library, and the deduced amino
acid sequence of 313 residues revealed that the mature form of P35
consists mainly of collagen- and fibrinogen-like domains. The latter
contained two potential Ca
-binding sites that may be
involved in carbohydrate binding. The overall sequence of P35 was
highly homologous to porcine ficolins
and
. Northern blots
of various human tissues showed that the major product of the
1.3-kilobase-long P35 transcript is expressed in liver. P35 enhanced
phagocytosis of Salmonella typhimurium by neutrophils,
suggesting an opsonic effect via the collagen region. P35 was found to
bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as
well as to neoglycolipids containing complex-type oligosaccharides
derived from glycoproteins, suggesting that P35 recognizes GlcNAc
residues such as those found in microbial glycoconjugates and
complex-type oligosaccharides. Therefore, P35 represents a new type of
GlcNAc-binding lectin with structural and functional similarities to
collectins involved in innate immunity.
Animal lectins are mainly involved in endocytosis, cell-cell
interactions, and host defense. Collectins (1, 2, 3, 4) are a group of
oligomeric C-type (Ca-dependent) (5) animal
lectins and include three types of serum lectins (mannose-binding
protein (MBP)(
)(6, 7) , conglutinin, and
CL-43(8) ) and two pulmonary surfactant proteins (SP-A and
SP-D(9) ). Their structures and functions are related to C1q, a
subcomponent of the first complement component (C1).
Collectins
possess a structural unit made up of three polypeptide chains, each
containing at least three domains. The first domain consists of an
NH-terminal sequence containing cysteine residues that make
it possible for structural units to form oligomers via disulfide bonds.
The second domain is a collagenous region consisting of
Gly-X-Y triplet repeats (where X and Y represent any amino acid). The third domain contains a
COOH-terminal sequence consisting of a carbohydrate recognition domain
(CRD) (10) that is also involved in Ca
binding. It has 14 invariant and 18 highly conserved residues
spread over
120 amino acids and is found in many animal
lectins(11, 12) .
Collectins play a role in the first line of host defense against microorganisms possessing certain oligosaccharides on their surface. Opsonization appears to be their common function, as is the case with MBP (13, 14) and conglutinin(15) , and may occur through the interaction of collagenous regions with the C1q receptor on phagocytes (16, 17) . MBP and conglutinin have been shown to bind to gp120 and gp160 of the envelope glycoproteins of human immunodeficiency virus type 1, respectively(18, 19) , thereby inhibiting virus infection of cells (20) or the interaction between gp160 and CD4(19) . In addition, MBP has the unique property of being able to activate the complement system(21, 22, 23, 24) , probably by utilizing a C1s-like serine protease termed MBP-associated serine protease(25, 26) , which is capable of cleaving C4 and C2, leading to the killing of bacteria or viruses by direct lysis or opsonization.
We describe here a novel type of human serum
Ca-dependent and GlcNAc-binding lectin with opsonic
activity. This lectin has a collagenous domain similar to that of
collectins. Unlike the CRD in collectins, however, it possesses a
fibrinogen-like domain that is probably involved in carbohydrate
binding.
Figure 2:
NH-terminal amino acid
sequence of P35 determined with a protein sequencer. The underlined sequences were used for the construction of two oligonucleotide
probes.
As shown in Fig. 1, P35 showed
a single band that was slightly larger than the MBP subunit under
reducing conditions. Under nonreducing conditions, it appeared as a
320-kDa moiety. These results suggest that P35 is composed of subunits
linked to form a homopolymer via disulfide bonds, as is the case with
MBP. The NH-terminal 43-amino acid sequence of P35
determined using a protein sequencer shows glycine residues at
intervals of two amino acids, suggesting a collagenous type of sequence (Fig. 2).
Figure 1: SDS-polyacrylamide gel electrophoresis of P35. MBP (lanes a) or P35 (lanes b) was subjected to SDS-polyacrylamide gel electrophoresis under reducing (left; 10% gel) or nonreducing (right; 5% gel) conditions. Proteins were stained with Coomassie Brilliant Blue R-250. Molecular size markers are indicated on both sides.
Figure 3:
cDNA
and deduced amino acid sequences of P35. The nucleotide sequence (upper) and the deduced amino acid sequence (lower)
are numbered. The leader sequence is singly underlined. The
NH-terminal sequence determined for the P35 protein is doubly underlined. Two potential N-glycosylation
sites (boxed) and two potential calcium-binding sites (dashed lines) are indicated.
By conducting a homology search, we found that P35 shows 77.4 and
75.4% homology to ficolins and
, respectively, at the amino
acid level (Fig. 4). Ficolin was first identified in porcine
uterus membranes and characterized as a transforming growth
factor-
-binding protein by Ichijo et al.(39) ,
although its physiological functions remain unknown. These
investigators isolated two closely related cDNA clones encoding two
proteins designated ficolins
and
, which have 83% homology
and which consist of collagen- and fibrinogen-like
domains(40) , as seen in P35.
Figure 4:
Comparison of amino acid sequences of P35
and porcine ficolins and
. The amino acid sequences of
ficolins
and
are according to Ichijo et
al.(40) . Identical amino acids in all proteins are shaded.
Figure 5: Northern blot of P35. A membrane filter containing mRNA from human tissues was hybridized with the P35 cDNA fragment corresponding to nucleotides 110-1080. Size markers are indicated on the left. There are four transcripts of 4.0, 3.2, 3.0, and 1.3 kb in liver and 1.4-kb transcripts in lung and placenta.
Figure 6:
Binding of P35 to neoglycoproteins and
natural glycoproteins. Neoglycoproteins and natural glycoproteins were
dot-blotted on an Immobilon-P membrane. The membrane was incubated with I-labeled P35 and then subjected to autoradiography for
24 h for detection of P35 binding. Details are described under
``Experimental Procedures'' and ``Results.'' OVA, ovalbumin; RN, RNase B; Fib,
fibrinogen; Fet, fetuin; AGP,
-acid
glycoprotein; Lac, lactose; Cel,
cellobiose.
We next performed a similar binding assay using neoglycolipids
containing neutral oligosaccharides derived from the above six
glycoproteins. Neoglycolipids were prepared by conjugating neutral
oligosaccharides derived from the glycoproteins to
dipalmitoylphosphatidylethanolamine. These conjugates were
chromatographed on a TLC plate, which was then overlaid with I-P35. As shown in Fig. 7, radiolabeled P35 bound
to neoglycolipids containing fully galactosylated bi-, tri-, and
tetraantennary complex-type oligosaccharides derived from fibrinogen,
fetuin, and
-acid glycoprotein (Fig. 7, lanes d-f, respectively). In addition, P35 bound to
neoglycolipids containing di-, mono-, and non-galactosylated
biantennary complex-type oligosaccharides of IgG (lane c).
However, P35 did not bind to those containing high mannose-type or
hybrid-type oligosaccharides derived from ovalbumin and RNase B (lanes a and b, respectively). The observation that
P35 bound to IgG oligosaccharides lacking terminal galactose residues
(bands indicated by arrows in lane c) and to
complex-type oligosaccharides with terminal galactose residues, but not
to high mannose-type or hybrid-type oligosaccharides with GlcNAc
residues in the trimannosyl core structures, suggests that P35
recognizes the GlcNAc residue that is next to galactose and that is
linked to the trimannosyl core in complex-type oligosaccharides.
Figure 7:
Binding of P35 to neoglycolipids derived
from glycoproteins. Neoglycolipids prepared from neutral
oligosaccharides of the six glycoproteins, as described under
``Experimental Procedures,'' were chromatographed on a TLC
plate. Lane a, ovalbumin; lane b, RNase B; lane
c, IgG; lane d, fibrinogen; lane e, fetuin; lane f, -acid glycoprotein. The plate was
overlaid with
I-labeled P35, subjected to autoradiography
for 24 h for detection of P35 binding (upper panel), and then
stained with orcinol reagent for oligosaccharide detection of the
neoglycolipids (lower panel). Arrows in lane c indicate a neoglycolipid lacking terminal galactose derived from
IgG.
The finding that the binding of P35 to glycoproteins and neoglycolipids was different, although both contain the same oligosaccharide structures, may be ascribed to a difference in clustering of the carbohydrate structures. On natural glycoprotein molecules, unlike neoglycoproteins, oligosaccharide chains are dispersed. However, the oligosaccharides of neoglycolipids are probably clustered on TLC plates coated with silica gel because of the hydrophobic nature of their lipid moiety. Therefore, it was suggested that P35 may prefer clustered GlcNAc residues and clustered complex-type oligosaccharide chains with GlcNAc residues linked to the trimannosyl core. The possibility still remains that oligosaccharides are hidden by the polypeptide moiety or by sialic acid residues linked to galactose residues of oligosaccharide chains in glycoproteins and that they are exposed in neoglycolipids in which the hindering moieties have been removed.
Figure 8:
P35
binding to GlcNAc-agarose or asialofetuin-Sepharose. One milliliter of
P35 (100 µg) was loaded onto a GlcNAc-agarose (A) or an
asialofetuin-Sepharose (B) column that had been equilibrated
with 50 mM Tris, 150 mM NaCl, and 10 mM CaCl, pH 7.8 (Tris buffer). After washing with Tris
buffer, elution of the GlcNAc-agarose column was carried out with Tris
buffer containing 150 mM GlcNAc. Elution of the
asialofetuin-Sepharose column was first carried out with Tris buffer
containing 150 mM Gal and then with Tris buffer containing 150
mM GlcNAc. Fractions collected from each column were subjected
to SDS-polyacrylamide gel electrophoresis, and the results are
shown.
Figure 9: Binding of P35 to S. typhimurium. S. typhimurium cells (TV119 (left) or LT2 (right)) were incubated with P35 or buffer alone as a control. In inhibition experiments with TV119, P35 was preincubated with GlcNAc or mannan. The bacteria were then exposed to anti-P35 monoclonal antibody 2F5 followed by fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulins. Fluorescence was recorded in arbitrary units on a logarithmic scale and is plotted against relative cell number.
Figure 10: Phagocytosis of S. typhimurium by PMN. S. typhimurium cells (TV119 or LT2) were incubated with GHBSS or GHBSS containing various amounts of P35 (2, 10, and 50 µg/ml). After washing, the bacteria were incubated with PMN to promote phagocytosis. Numbers of bacteria ingested by PMN were determined by microscopy. The phagocytic index is the number of targets ingested by 100 PMN. Error bars indicate the standard deviation of six independent assays. P35 significantly enhanced uptake of TV119 by PMN (*, p < 0.05;**, p < 0.01).
We isolated and characterized a human serum lectin tentatively named P35 that is capable of binding to yeast mannan-Sepharose and elutes with GlcNAc. Binding studies with neoglycoproteins and neoglycolipids indicate that P35 does not bind to mannose-BSA or to high mannose-type or hybrid-type oligosaccharides, although it binds to GlcNAc in GlcNAc-BSA and complex-type oligosaccharide chains with GlcNAc residues linked to the trimannosyl core. In experiments with mannan-Sepharose, P35 may not have bound to the mannose residue but to GlcNAc-containing oligosaccharides present as contaminants in the yeast mannan used since yeast mannan itself does not contain this type of oligosaccharide(41) . The binding specificity of P35 for GlcNAc was also confirmed by affinity chromatography using GlcNAc-agarose and asialofetuin-Sepharose. Purified P35 was able to bind to both columns and eluted with GlcNAc (Fig. 8).
P35, MBP, and conglutinin can be classified as
serum lectins specific for GlcNAc. Based on binding studies with
neoglycolipids, it has been shown that MBP(42, 43) ,
conglutinin(36) , and P35 recognize a GlcNAc next to a terminal
galactose in oligosaccharides derived from IgG when this terminal
galactose is missing. Unlike P35, MBP and conglutinin are unable to
bind to GlcNAc when an adjacent terminal galactose is present. MBP and
conglutinin bind to mannose in oligosaccharides derived from
glycoproteins such as RNase B and ovalbumin. In contrast, P35 is unable
to bind to high mannose-type or hybrid-type oligosaccharides or to
mannose-BSA. The lack of binding to hybrid-type oligosaccharides
bearing a GlcNAc1-Man group may be due to steric hindrance by the
outer chain on the Man
1-6Man
1 side. It can be concluded
that P35 shows binding specificity toward GlcNAc and that, unlike MBP
and conglutinin, it does not recognize mannose.
The precise mechanism by which P35 is able to bind to neoglycolipids is unknown, as is the reason why it does not bind to glycoproteins even though both contain the same oligosaccharide structure. As described under ``Results,'' however, P35 may prefer clustered GlcNAc residues and clustered complex-type oligosaccharide chains with GlcNAc residues linked to the trimannosyl core. This preference is in accord with the binding of P35 to microorganisms that have a large number of nonreducing terminal GlcNAc residues exposed on their surface, as is the case with bacteria of S. typhimurium strain TV119. On the other hand, P35 was unable to bind to LT2, a smooth-type strain of S. typhimurium that possesses additional O-polysaccharides. These extra O-polysaccharides might render GlcNAc inaccessible to P35 due to steric hindrance. Therefore, we could not exclude the possibility that the GlcNAc residues of the glycoprotein oligosaccharide chains may be hidden by the polypeptide portion or by sialic acid residues that are linked to galactose residues on the these oligosaccharides, resulting in the prevention of a P35 interaction.
By searching for sequence homology
in a protein data base, we found that the NH-terminal
six-amino acid sequence of P35 completely coincides with that of a
36-kDa protein that was identified as one of three soluble class I
human leukocyte antigen proteins in human plasma by immunoprecipitation
and immunoblotting using anti-human leukocyte antigen monoclonal
antibodies(44) . Of the three proteins, the
NH
-terminal amino acid sequences of two species were
identical to that of cellular human leukocyte antigen, whereas no
homology was found for the other 36-kDa protein. It is possible that
the 36-kDa protein is identical to P35 and was obtained because of its
ability to bind to IgG through complex-type oligosaccharides.
cDNA
analysis revealed that P35 and porcine ficolin are highly homologous
and share collagen- and fibrinogen-like domains. Two types of ficolin
with sequence homology, ficolins and
, have been identified
from a porcine uterus cDNA library. The distribution of ficolin in
various human tissues has been studied using porcine ficolin probes and
mRNAs from these tissues; and ficolin
was shown to be present in
lung and placenta, and ficolin
in skeletal muscle (40) .
With Northern blot analysis, we showed that the major P35 transcript of
1.3 kb is expressed in liver. This transcript seems to correspond to
P35. Three larger signals that were also detected in liver probably
correspond to products of alternative splicing because they were found
in the cDNA sequences, and Northern blotting with the intron sequences
as a probe showed that the probes were reacted with these three bands,
but not with the 1.3-kb band. (
)Lung and placenta expressed
cross-hybridized messages 1.4 kb in length, which is slightly longer
than that in liver, suggesting that these two tissues produce
P35-related proteins that may differ in amino acid sequence from the
one expressed in liver. It is possible that P35 and P35-related protein
might be human analogues of the two types of porcine ficolin.
P35
and collectins share structural and functional similarities. They are
both Ca-dependent lectins as well as hybrid proteins
that have collagenous domains with either a fibrinogen-like domain (in
P35) or a CRD (in collectins). Amino acid sequences in the CRD
responsible for calcium and carbohydrate binding have been thoroughly
investigated(11, 12) . Although there is no sequence
homology between the CRD and the fibrinogen-like domain, P35 has
potential calcium-binding sites within the latter. It is therefore
reasonable to expect that carbohydrate-binding sites also reside in
this domain. It has been reported that fibrinogen-like domains occur in
certain proteins such as tenascin and cytotactin (45, 46) and the scabrous protein of Drosophila
melanogaster(47) ; however, their functions in these
proteins are not yet fully understood. This is the first report
demonstrating that one possible function of the fibrinogen-like domain
might be to bind to carbohydrates in a calcium-dependent manner.
P35 has been demonstrated to bind to TV119, an Ra chemotype strain of S. typhimurium bearing rough core polysaccharides containing a terminal GlcNAc. Binding of P35 to TV119 was carbohydrate-specific and mediated enhancement of phagocytosis by PMN. These activities were not directed to LT2, a smooth-type strain of S. typhimurium. O-Polysaccharides in LT2 might render P35 inaccessible to GlcNAc due to steric hindrance. Collagen domains in collectins are crucial for exhibiting opsonic activities. Upon binding to their ligands, collectins interact with the C1q receptor on phagocytes, resulting in enhancement of phagocytosis. Since P35 is also a lectin containing a collagen-like domain structure, it is possible that in P35, this domain binds to the C1q receptor on PMN.
In conclusion,
P35 is a human serum Ca-dependent lectin specific for
GlcNAc. It exhibits a high structural similarity to porcine ficolin and
possesses both collagen- and fibrinogen-like domains. P35 also
resembles collectins in that the collagen-like domains in both are
presumably involved in opsonic activities toward microorganisms.
Instead of the CRD found in collectins, however, P35 has a
fibrinogen-like domain that could be responsible for carbohydrate
binding. P35 is therefore a novel type of serum
Ca
-dependent and GlcNAc-binding lectin with hybrid
domains that are probably involved in innate immunity.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49353[GenBank].