(Received for publication, October 23, 1995)
From the
Gene fusions encoding the membrane anchor region of yeast
1,2-mannosyltransferase (Mnt1p) fused to human
1,4-galactosyltransferase (Gal-Tf) were constructed and expressed
in the yeast Saccharomyces cerevisiae. Fusion proteins
containing 82 or only 36 N-terminal residues of Mnt1p were produced and
quantitatively N-glycosylated; glycosyl chains were shown to
contain
1,6-, but not
1,3-mannose determinants, a structure
typical for an early Golgi compartment. A final Golgi localization of
both fusions was confirmed by sucrose gradient fractionations, in which
Gal-Tf activity cofractionated with Golgi Mnt1p activity, as well as by
immunocytological localization experiments using a monoclonal
anti-Gal-Tf antibody. In an in vitro Gal-Tf enzymatic assay
the Mnt1/Gal-Tf fusion and soluble human Gal-Tf had comparable K
values for UDP-Gal (about 45
µM). To demonstrate in vivo activity of the
Mnt1/Gal-Tf fusion the encoding plasmids were transformed in an alg1 mutant, which at the non-permissive temperature transfers
short (GlcNAc)
glycosyl chains to proteins. Using specific
lectins the addition of galactose to several yeast proteins in
transformants could be detected. These results demonstrate that Gal-Tf,
a mammalian glycosyltransferase, is functional in the molecular
environment of the yeast Golgi, indicating conservation between yeast
and human cells. The in vivo function of human Gal-Tf
indicates that the yeast Golgi is accessible for UDP-Gal and suggests
strategies for the construction of yeast strains, in which desired
glycoforms of heterologous proteins are produced.
N-Glycosyl chains of glycoproteins can have a great
variety of structures in mammalian cells, while they are built
relatively simply in lower eukaryotes, such as the yeast Saccharomyces cerevisiae. Nevertheless, in both types of cell
the dolichol-linked precursor is identical and the selection of
asparagine attachment sites proceeds similarly. Yeast core N-glycosyl chains can be extended by the addition of >100
mannose units(1) ; in contrast, a multitude of enzymes in
mammalian cells can process the core to create diverse N-glycosyl chains containing various sugars(2) . The
complex type of N-glycosyl chains in mammalian cells is
synthesized after trimming of the core unit by the sequential addition
of GlcNAc, Gal, and NeuAc. Galactose addition in human cells is
mediated by
UDP-galactose:N-acetyl-D-glucosaminyl-glycopeptide
4-D-galactosyltransferase (EC 2.4.1.38) (Gal-Tf), (
)an enzyme anchored in the Golgi membrane, which uses
UDP-Gal as substrate and GlcNAc on glycosyl structures as
acceptors(3, 4) . In its secreted soluble form, which
lacks a membrane anchor region, Gal-Tf functions as lactose synthase,
while a third role of Gal-Tf, correlated with its localization on the
cell surface, is to mediate adhesion processes(5) .
The
targeting of Gal-Tf and other glycosyltransferases to the Golgi
requires specific protein sequences in these proteins, as well as
cellular components (reviewed in (6) ). The membrane anchor
region of Gal-Tf contains essential sequences for its targeting to the trans-Golgi(7, 8, 9, 10) .
Similarly, Golgi localization of yeast glycosyltransferases appears to
be mediated by their membrane anchor regions(11, 12) .
It has been shown that the transmembrane domain of yeast
1,2-mannosyltransferase (Mnt1p), which extends O-glycosyl
chains in the Golgi, is necessary for Golgi localization(11) .
1,3-mannosyltransferase (Mnn1p) contains separable Golgi
localization signals within both the transmembrane and lumenal
domains(12) . Recently, it has been shown that the membrane
anchor region of a mammalian glycosyltransferase, rat
2,6-sialyltransferase, is able to target a reporter protein to the
yeast Golgi, indicating conservation of targeting mechanisms between
lower and higher eukaryotes(13) . The transmembrane
domain-mediated localization of yeast glycosyltransferases appears
distinct from other Golgi proteins, whose targeting depends on
Y/F-X-Y/F sequences in their cytoplasmic tails(14) .
It has been shown that the production of mammalian
glycosyltransferases in heterologous host cells can alter the
glycosylation pattern of such cells. Several glycosyltransferases have
been isolated by heterologous expression in Chinese hamster ovary cells
(reviewed in Refs. 15, 16). In mouse cells transfected with the human
gene encoding 1,2-fucosyltransferase blood group H determinants
are synthesized (17) . An Arabidopsis thaliana mutant
defective in N-acetylglucosaminyltransferase I can be
complemented by the human cDNA for this enzyme(18) . Recently,
it has been reported that mammalian glycosyltransferases can be
produced in the yeast S. cerevisiae in an active
form(19, 20, 21) , but an effect on yeast
glycosyl structures was not observed. Such a modifying effect would
have been expected only if the respective glycosyltransferase were
synthesized at sufficient levels in the proper organelle (such as the
Golgi) in yeast cells, on condition that acceptor structures and
activated sugars were present(22) .
In the present study we demonstrate that a fusion of the membrane anchor region of yeast Mnt1p to soluble human Gal-Tf can be produced at high levels and targeted to the Golgi in yeast cells. We present evidence that the Gal-Tf fusion is enzymatically active and in vivo is able to add galactose residues to acceptor structures in an alg1 mutant. The function of the Gal-Tf fusion in the molecular environment of the yeast Golgi suggests that essential Golgi structures and constituents are conserved between lower and higher eukaryotes and specifically indicates that UDP-Gal is available in the lumen of the yeast Golgi. We propose that the modification of glycosyl structures by the action of heterologous glycosyltransferases may be used as a strategy to construct new yeast host strains that secrete desired glycoforms of applied proteins(23) .
The BamHI-HindIII fragment carrying the MNT1::Gal-Tf expression unit was inserted into the BamHI and HindIII sites of pJDB207(28) , thereby placing the 3` end of the expression unit next to the 2-µm FLP terminator following the HindIII site. The final vector was designated phMGT1 (Fig. 1).
Figure 1: Structures of MNT1/Gal-Tf expression units. The MNT1 and Gal-Tf gene segments are indicated (open boxes); the GAL10 promoter is represented by the arrow. Sequences at the fusion junctions (dashed line) of the encoded proteins are shown along with residue numbers of the original proteins. The position of the deletion in the phMGT3-encoded fusion is indicated. B, BamHI; R, EcoRI; H, HindIII.
A deleted version of
phMGT1 was constructed that only contains residues 1-28 and
75-82 of Mnt1p. First, the GAL10 promoter was removed
from pGTÜ1 by cutting with BamHI and NcoI, fill-in of ends with Klenow polymerase, and religation
(the NcoI site was regenerated during the ligation). The large EcoRI fragment (containing vector and N-terminal MNT1 sequences) and the 1.0-kilobase EcoRI fragment
(containing MNT1 and Gal-Tf sequences) of the resulting
plasmid pGTÜ were ligated. In pMNT1
RI MNT1 anchor and Gal-Tf coding regions were fused in-frame.
Because of 2 EcoRI sites corresponding to amino acids 27/28
and 73/74 of MNT1 this procedure generated a significant
deletion removing 2 amino acids of the membrane anchor (residues 29 and
30) and most residues of the stem region. The NcoI-HindIII fusion fragment was joined to the GAL10 promoter fragment and reinserted into pJDB207, as
described above, to construct phMGT3 (Fig. 1).
To monitor the in vivo activity of Gal-Tf,
transformants of strain ATS594-1B were grown at 26 °C to an
OD of 0.5-0.8 in low sulfate medium containing
2% galactose and 0.2% glucose(13) . 10 OD
units were harvested and resuspended in 1 ml of low sulfate
medium lacking glucose and containing 0.24% bovine serum albumin. After
preincubation at 37 °C, 26 µl of Tran
S-label (ICN
Biomedicals, Inc.) (300 µCi) were added and the sample was
incubated further at 37 °C for 60 min. 20 µl of 50
chase solution (50 mM (NH
)
SO
, 250 mML-methionine, 50 mML-cystein) was
added and the incubation was continued for 15 min. Cells were harvested
by centrifugation (4000 rpm/5 min), washed twice with 2 ml of ice-cold
10 mM NaN
, 10 mM dithiothreitol and
resuspended in 450 µl of LIP buffer (50 mM Tris-HCl, pH
7.4, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA). Cells
were frozen at this stage at -80 °C prior to further
processing. Phenylmethylsulfonyl fluoride was added to 1 mM and cells were thawed on ice and disrupted with an equal volume of
glass beads on a Braun homogenizer (2
2 min). Following
inactivation of Gal-Tf at 95 °C (5 min) the extracts were placed on
ice and centrifuged 2 min at 5000 rpm. The supernatant was transferred
to a new vial, 50% (NH
)
SO
was added
to a total volume of 1.5 ml and allowed to stand on ice for a minimum
of 3 h. The suspension was centrifuged for 5 min at 14,000 rpm (4
°C), after which the supernatant was discarded and the protein
pellet was washed with 500 µl of ice-cold 25%
(NH
)
SO
. The pellet was solubilized
in 50-100 µl of 0.2% Triton X-100 by vortexing and LIP buffer
was added to 1.5 ml. The solution was precleared by the addition of 30
µl of 50% Sephacryl S-200 (Pharmacia) and mild agitation at 4
°C for 1-2 h, followed by centrifugation at 14,000 rpm (2
min). The supernatant was divided in 3 portions of 500 µl, to which
were added either 40 µl of 20% ConA-Sepharose 4B (Sigma), or 40
µl of 50% agarose-coupled wheat germ agglutinin (WGA) (Sigma), or
40 µl of 50% agarose-coupled agglutinin RCA
(RCA)
(Sigma). Following mild agitation overnight at 4 °C the suspensions
were centrifuged (14,000 rpm/4 °C), the supernatants discarded, and
the pellets were washed twice with LIP buffer and twice with
phosphate-buffered saline. Pellets were resuspended in Laemmli sample
buffer and proteins were separated by SDS-PAGE.
Figure 2:
Production of Mnt1/Gal-Tf fusions by yeast
transformants. A, extracts of transformants carrying phMGT1 (lane 1), phMGT3 (lane 2), and pJDB207 (lane
3) were analyzed by immunoblotting using a monoclonal anti-Gal-Tf
antibody. B, transformants were labeled by
[S]methionine and proteins in cell extracts were
immunoprecipitated using anti-Gal-Tf antibody. Immunoprecipitates were
analyzed by SDS-PAGE followed by autoradiography. Extracts of a phMGT1
transformant (lanes 4 and 5) and of a phMGT3
transformant (lanes 6 and 7) are shown; samples
loaded in lanes 5 and 7 had been treated by PNGase F.
The apparent molecular masses of prestained standard proteins (Sigma)
are indicated.
Gal-Tf contains a single potential site for N-glycosylation, which is glycosylated in human
cells(33) . To test the glycosylation status of the
yeast-produced Gal-Tf fusion we treated immunoprecipitates of S-labeled transformants with PNGase F. Gal-Tf was
immunoprecipitated using an anti-Gal-Tf antibody; the precipitates were
separated by SDS-PAGE and gel patterns were visualized by
autoradiography (Fig. 2B). PNGase F-treated Mnt1/Gal-Tf
migrates slightly faster than untreated Mnt1/Gal-Tf, an effect that is
especially apparent for the phMGT3-encoded Gal-Tf (Fig. 2B, compare lanes 4, 5 and 6,
7). We estimate that N-glycosylation increases the
molecular mass of the yeast-produced Mnt1/Gal-Tf fusion protein by
about 3 kDa.
The small size of its N-glycosyl chain was
consistent with only core N-glycosylation of the Gal-Tf
fusion. To test if the fusion protein nevertheless had acquired typical
Golgi modifications, sequential immunoprecipitations were carried out
by first using anti-Gal-Tf antibody(29) , followed by either
anti-1,6-mannose or anti-
1,3-mannose antibodies; in addition,
in the second step aliquots were immunoprecipitated with concanavalin A
or, as a control, with anti-Gal-Tf antibody. Similar results were
obtained for the phMGT1- and phMGT3-encoded fusion proteins (Fig. 3). The anti-
1,6-mannose antibody and concanavalin A
were similarly efficient as the anti-Gal-Tf antibody to precipitate the
Gal-Tf fusions (Fig. 3, lanes 2, 3, 5 or lanes 7,
8, 10); however, no immunoprecipitation occurred with the
anti-
1,3-mannose antibody. These results indicate that the Gal-Tf
fusion proteins have quantitatively obtained the
1,6-mannose
modification in the Golgi; the absence of
1,3-modification
indicates that the fusion proteins have not reached distal Golgi
compartments.
Figure 3:
Golgi modification of Mnt1/Gal-Tf fusions.
Immunoanalyses of yeast extracts were performed as in Fig. 2B. Immunoprecipitates obtained with anti-Gal-Tf
antibody (lanes 1 and 6) were solubilized, and
aliquots were again immunoprecipitated with anti-Gal-Tf antibody (lanes 2 and 7), anti-1,6-mannose antibody (lanes 3 and 8), anti-
1,3-mannose antibody (lanes 4 and 9), or concanavalin A (lanes 5 and 10). Extracts of the phMGT3 transformant (lanes
1-5) and the phMGT1 transformant (lanes 6-10)
were analyzed.
If the Mnt1/Gal-Tf fusions had inserted into yeast organelle membranes as the authentic Mnt1 and Gal-Tf proteins(4, 11, 34) , which are type II membrane proteins, it could be expected that they are refractory to the action of proteases. To test this notion crude extracts of phMGT1 and phMGT3 transformants were treated with proteinase K in the presence and absence of Triton X-100; subsequent to the treatments the Gal-Tf fusion protein was analyzed on immunoblots using the anti-Gal-Tf antibody. It is demonstrated in Fig. 4that the phMGT1- and phMGT3-encoded fusion proteins showed identical properties in this assay: the Gal-Tf fusion protein was only stable in the presence of protease, if no detergent was added (Fig. 4, lanes 3 and 7), while degradation occurred in its presence (Fig. 4, lanes 4 and 8). The shortened Gal-Tf fusion encoded by phMGT3 appeared somewhat more resistant in this assay, suggesting that the deleted region compared to phMGT1 contains sequences especially sensitive to proteolysis. The protease protection experiments clearly indicate that the Mnt1/Gal-Tf fusion proteins reside in a closed vesicular compartment.
Figure 4: Protease protection of Mnt1/Gal-Tf fusions. Extracts of a phMGT1 transformant (lanes 1-4) and a phMGT3 transformant (lanes 5-8) were not pretreated (lanes 1, 2, 5, and 6) or pretreated with proteinase K (lanes 3, 4, 7, and 8) in the absence (lanes 1, 3, 5, and 7) or presence of Triton X-100 (lanes 2, 4, 6, and 8).
Figure 5:
Mnt1/Gal-Tf and marker proteins in cell
fractions obtained by sucrose gradient centrifugation. Organelles of a
phMGT1 transformant, prepared by method A (see ``Experimental
Procedures''), were separated on a sucrose density gradient, and
gradient fractions were analyzed for the presence of Mnt1/Gal-Tf and
marker proteins by enzymatic activity tests. Gal-Tf, Gal-Tf
activity of Mnt1/Gal-Tf fusion; oxidoreductase,
NADPH-dependent cytochrome c oxidoreductase; oxidase,
cytochrome c oxidase; mannosidase,
-mannosidase.
Figure 6: Mnt1/Gal-Tf and marker proteins in cell fractions obtained by sucrose gradient centrifugation. Cell extracts of a phMGT1 transformant, prepared by method B (see ``Experimental Procedures''), were separated on a sucrose density gradient and analyzed as described in the legend to Fig. 5. The relative ATPase protein level is indicated (the maximal level in fraction 17 was arbitrarily assigned the value 1.0).
To confirm Golgi localization of the Mnt1/Gal-Tf fusion, immunocytological localization experiments using the monoclonal anti-Gal-Tf antibody were carried out. phMGT1-transformant cells, but not control transformants carrying pJDB207, displayed a punctuated immunofluorescent staining characteristic of the yeast Golgi(11) . No fluorescence was detected in the vacuole, which was clearly visible by differential interference contrast microscopy or in association with the nucleus, which was stained by DAPI (Fig. 7). Thus, the fractionation and immunocytological localization experiments indicate that the Mnt1p/Gal-Tf fusions encoded by the phMGT vectors are located in the Golgi and not in other cellular organelles.
Figure 7: Immunocytological localization of Gal-Tf. Transformants carrying phMGT1 or pJDB207 were treated with anti-Gal-Tf antibody, which was reacted with dichlorotriazinyl aminofluorescein-coupled anti-mouse antibody; in addition, cells were treated with DAPI. The appearance of cells as detected by differential-interference-contrast microscopy (DIC), DAPI-fluorescence, and indirect immunofluorescence (for Gal-Tf) was examined.
Figure 8:
In vitro Gal-Tf activity. The
enzymatic activity of 0.25 µg of Mnt1/Gal-Tf in cell extracts of a
BJ1991[phMGT1] transformant () and of soluble human
Gal-Tf (+) were determined with ovalbumin as acceptor
protein.
In transformants carrying control vector pJDB207 several proteins were precipitated with ConA and WGA (Fig. 9, lanes 1 and 2), indicating mannose and GlcNAc residues; the high level of mannosylated proteins suggests that the alg1 mutation in strain ATS594-1B is leaky at the non-permissive temperature (25) . No proteins could be precipitated with lectin RCA, demonstrating the absence of terminal galactose in ATS594-1B[pJDB207] transformants (Fig. 9, lane 3). In contrast, several proteins could be detected in transformants carrying plasmid phMGT1 (Fig. 9, lane 6) (similar results were obtained for the phMGT3 transformant). Although in this experiment Gal-Tf was heat-inactivated, we considered the possibility that GlcNAc-carrying proteins only became galactosylated because of the liberation of Gal-Tf and yeast proteins during cell breakage. In a control experiment we added 100 mM GlcNAc before cell breakage, whose presence competes with the transfer of galactose to ovalbumin in the in vitro Gal-Tf assay (Table 2). Since proteins still became efficiently galactosylated after this treatment (Fig. 10) this process must have occurred in the intact cell, i.e. during the transit of proteins via the Golgi. These results show that expression of the MNT1::Gal-Tf gene fusion is able to allow the galactosylation of a number of secreted yeast proteins. The presence of non-galactosylated proteins carrying GlcNAc residues (Fig. 9, compare lanes 5 and 6) may be due to proteins, whose transit from the endoplasmic reticulum to the Golgi does not occur in the absence of normal core glycosylation.
Figure 9:
In vivo Gal-Tf activity. The alg1 strain ATS594-1B carrying control vector pJDB207 (lanes 1-3) or phMGT1 (lanes 4-6) was S-labeled at the non-permissive temperature. Glycosylated
proteins were precipitated with immobilized lectins ConA (lanes 1 and 4), WGA (lanes 2 and 5), or RCA (lanes 3 and 6) and separated by SDS-PAGE, followed
by autoradiography.
Figure 10: In vivo Gal-Tf activity in the presence of GlcNAc. Protein glycosylation of strain ATS594-1B[phMGT1] was determined essentially as described in the legend to Fig. 9, but by using cells broken in the presence of 100 mM GlcNAc. For lectin-precipitations ConA (lane 1), WGA (lane 2), and RCA (lane 3) were used.
We report here that a mammalian glycosyltransferase, human Gal-Tf, can be targeted to the yeast Golgi, where it is inserted in authentic orientation and attains an active conformation. We present evidence that the yeast-produced Gal-Tf is able to add galactose residues to the truncated glycosyl chains of alg1 mutants. These results have implications regarding the basic mechanisms of targeting and function of glycosyltransferases, as well as for the use of yeast as a host for the production of heterologous proteins.
We
previously found that the membrane anchor region of rat
2,6-sialyltransferase is able to direct the localization of a
reporter protein to the yeast Golgi(13) . Likewise, a fusion of
the human Gal-Tf membrane anchor region to invertase was targeted to
the yeast Golgi, although production levels were low. (
)Initial experiments on expression of the gene encoding
full-length human Gal-Tf in yeast, however, were unsuccessful, because
Gal-Tf was not synthesized in spite of a considerable transcript
level(19) . Therefore, we chose to express a gene fusion
encoding the membrane anchor region of a yeast glycosyltransferase,
Mnt1p, fused to the soluble form of Gal-Tf (plasmid phMGT1). This gene
fusion is expressed in yeast at high transcript levels and leads to
high intracellular levels of Gal-Tf enzymatic activity(19) . We
had speculated that high transcript levels may be caused by a
``downstream activating sequence'' present in MNT1;
if such a sequence exists it does not appear to be situated in the
region encoding residues 29-74, because a deletion of this region
(in phMGT3) does not affect levels of Gal-Tf synthesis. The membrane
anchor region of Mnt1p has been shown previously to direct a reporter
enzyme to the yeast Golgi(11) . In the present study we confirm
and extend these results by demonstrating that the first 28 N-terminal
residues of Mnt1p encompassing the cytoplasmic tail, the membrane
anchor region plus 8 non-contiguous residues of the stem region are
sufficient to achieve Golgi localization. This result indicates that
lumenal sequences adjacent to the transmembrane region, as in
Mnn1p(12) , do not contribute to Golgi targeting of Mnt1p.
Golgi localization was proven by the presence of
1,6-mannose
determinants on Gal-Tf, a modification occurring in the early
Golgi(1) . Because some secreted proteins only temporarily
reach the Golgi before being retrieved to a preceding compartment (30) we demonstrated in two different types of sucrose
gradients that Gal-Tf comigrates with Golgi marker proteins, but not
with markers specific for other organelles. Since Gal-Tf did not
receive
1,3-mannose determinants it appears that its final
localization is in the cis- or medial-Golgi, but not
the trans-Golgi(1) . Thus, besides the already known
targeting mechanism using Y/F-X-Y/F sequences in the
cytoplasmic tail(14) , yeast appears to use a second mechanism
independent of such sequences, such as used by Mnt1p, Mnn1p, and the
heterologous sialyltransferase membrane
anchor(11, 12, 13) .
The function of a
mammalian membrane anchor region and, as we report here, the function
of a human glycosyltransferase in the molecular environment of the
yeast Golgi demonstrates that essential features affecting the
localization and function of glycosyltransferases are conserved between
lower and higher eukaryotes. Thus, the function of Gal-Tf in yeast does
not require auxiliary proteins present only in human cells. In the in vitro assay K values for UDP-Gal of
yeast-produced and human Gal-Tf were identical; the lowered V
values may be due to the lowered reactivity of
the membrane anchor-coupled (yeast) Gal-Tf compared to free (human)
Gal-Tf with respect to the ovalbumin acceptor in this assay. The
observed in vivo activity of Gal-Tf was unexpected in spite of
the presence of UDP-Gal and the alg1 acceptor structures in
the transformants, because yeast does not add galactose to proteins and
a transport system to import UDP-Gal in the Golgi lumen is not known
and does not seem necessary(22) . Our results imply that
UDP-Gal is present in the lumen of the Golgi; this could occur by the
action of a UDP-Gal transporter in the Golgi membrane, which functions
analogous to the GDP-Man carrier as an antiport system exchanging
lumenal UMP with cytoplasmic UDP-Gal. The latter mechanism appears
possible, because a UDPase activity that generates UMP from UDP, is
known to be present in yeast(35) . However, the existence of a
Golgi UDP-Gal import system remains to be demonstrated directly; such
studies also may reveal, if UDP-Gal co-utilizes existing transporters
for GDP-Man or, possibly, UDP-Glc(NAc).
The use of a alg1 mutant allowed the demonstration of in vivo activity of
Gal-Tf by providing glycosyl acceptors of the structure
GlcNAc-1,4-GlcNAc(25) . The temperature sensitivity of
this strain was not remedied by Golgi galactosylation, most likely
because the short N-glycosyl chains prevent transit of those
proteins from the endoplasmic reticulum to the Golgi, whose folding
depends on more extended glycosyl chains. However, many alg or mnn mutants are known, in which a variety of incomplete
glycosyl chains are produced; based on the principle shown in this
study such mutants may be used as hosts for the synthesis of
heterologous glycosyltransferases in an attempt to alter glycosyl
structures. It also appears feasible to simultaneously produce more
than one glycosyltransferases in one strain, such as to generate more
complex glycoforms in vivo. It will be an even greater
challenge to add synthesis reactions for activated sugars not present
in yeast, such as for the synthesis of CMP-NeuAc. We anticipate that
reconstitution of glycosylation pathways in yeast will on the one hand
help clarify the biology of mammalian glycosylation reactions. On the
other hand new host strains will be generated that will allow the
production of heterologous proteins, whose glycosyl chains are modified
by the addition of, for example, terminal Gal residues. These could be
initially further modified in vitro by the addition of sialic
acid (36) with the goal to shield mannose residues that are
targets for mannose-binding proteins and specific
antibodies(23) .