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INTRODUCTION |
The sialic acids (Sia)1
are a family of 9-carbon-carboxylated sugars containing nearly 50 members that are derivatives of N-acetylneuraminic acid
(Neu5Ac), N-glycolylneuraminic acid, and deaminoneuraminic acid
(2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid) (1). In most cases, Sia is present as a monomeric residue at the
non-reducing terminal position of glycan chains on glycoproteins and
glycolipids. By virtue of their net negative charge at physiologic pH,
Sia serve as mediators of ligand-receptor and cell-cell interactions (2). Sia are critically important because gene disruption of a
UDP-GlcNAc-2-epimerase/ManNAc kinase, an enzyme responsible for
biosynthesis of Sia, is lethal in embryonic mice (3).
Occasionally, Sia are linked to each other to form a polymerized
structure, polysialic acid (polySia). There is a large diversity in the
polySia structure arising from diversity in the sialic acid components
(Neu5Ac, N-glycolylneuraminic acid, and deaminoneuraminic acid) and in the intersialyl linkages
(
2
5O-glycolyl,
2
8,
2
9, and
2
8/9) (4).
2
8-Linked polySia structures with a
degree of polymerization (DP) ranging from 8 to 200 sialyl residues
occur in capsular components of neuroinvasive bacteria, such as
Escherichia coli K1 (5) and Neisseria
meningitidis group B (6), and in glycoproteins of fish eggs (7, 8) and mammalian brains (4, 9, 10), although it is not found in natural
glycolipids. In mammals, neural cell adhesion molecules (NCAM) (4, 9,
10) and the
-subunit of the voltage-sensitive sodium channel (11)
are the only two polySia-containing glycoproteins thus far identified.
Functions of the polySia chain in NCAM have been well studied. PolySia
NCAM is expressed in embryonic brains, which are undergoing neural
differentiation, but greatly decreases in the adult brain. During mouse
brain development, the polySia structure is converted to di/oligoSia in
NCAM, whereas NCAM expression remains unchanged (4, 10, 12). PolySia
NCAM was recently observed in hippocampus (13, 14) and hypothalamic
nuclei (15, 16) in adult brain where neurogenesis is ongoing. PolySia
NCAM is involved in neurite cell migration, axonal growth and path finding, synaptogenesis, and synaptic functions associated with learning and memory and circadian rhythm (4, 10). PolySia NCAM is also
expressed in several human cancer cells such as neuroblastoma (17),
Wilms' tumor (18), and small cell lung carcinoma (19) and is
widely recognized as a tumor marker (4). In tumor cells, polySia has an
important role in metastasis via an anti-adhesive effect between
cell-cell and cell-matrix adhesions (20, 21).
An intermediate size of sialyl chains between monoSia and polySia
occurs extensively in various glycoproteins in mammals (12, 22, 23,
25-27). This class of sialyl groups consists of diSia and oligoSia
with up to seven Sia residues and is present in functionally important
glycoproteins, such as adiponectin, which is involved in energy
consumption in obesity; CD166, which is involved in neurite extension
and T cell activation; and integrin, which is involved in cell-matrix
interactions. The study of these novel structures is possible because
of the recently developed, highly sensitive detection techniques (12,
28, 29) including fluorometric high performance liquid
chromatography and anti-di/oligo/polySia antibodies with defined
specificity. Our search for di/oligo/polySia-containing glycoproteins
in several cell lines using these methods revealed that COMMA-1D
cells, which derive from mammary epithelial cells, contain high amounts
of internal NeuAc (8-O-substituted Neu5Ac) residues (30).
This preliminary finding led us to search for polySia-containing
glycoprotein(s) in milk. We have now identified such glycoproteins in
human milk. In this study, we demonstrate that CD36 in human milk is a
new polySia-containing glycoprotein using both immunochemical and
chemical methods. This is the first report of the presence of
polySia-containing glycoprotein other than NCAM in non-neural tissues.
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EXPERIMENTAL PROCEDURES |
Materials--
Human colostrums (4-7 days after birth) and
mature milk (17-181 days after birth) were kindly provided by Dr.
Atsuo Urisu (Fujita Health University, Graduate School of Medicine,
Aichi, Japan) and Snow Brand Milk Products Co. Ltd. (Tokyo, Japan). All of the mothers gave informed written consent before inclusion in this
study. Mouse milk (3-8 day after birth) was obtained from 10-15-week-old ddY mice (SLC, Shizuoka, Japan). Embryonic pig brains were purchased from Tokyo Shibaura Zouki, Co. Ltd. (Tokyo, Japan). Endo-N-acylneuraminidase (Endo-N) was prepared from
bacteriophage K1F as described previously (31). Monoclonal antibody
(mAb), mAb 12E3 (IgM) recognizing oligo/polyNeu5Ac with the DP
5
and mAb S2-566 (IgM) specifically recognizing the Neu5Ac
2
8
Neu5Ac
2
3Gal sequence, were generously gifted from Dr.
Tatsunori Seki (Juntendo University School of Medicine, Tokyo,
Japan) and Dr. Koichi Furukawa (Nagoya University School of
Medicine, Nagoya, Japan), respectively. These antibodies were purified
as described previously (22). MAb 735 (IgG) recognizing polySia with DP
11 and mAb OL28 (IgM) recognizing oligo/polySia with DP
4 were kindly provided by Dr. Rita Gerady-Schahn (Medizinische Hochschule, Hannover, Germany) and Dr. Karen Colley (University of Illinois School
of Medicine), respectively. Rabbit polyclonal anti-CD36 antibodies
(anti-CD36) were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Anti-human NCAM antibody (2120), which also recognizes pig
NCAM, was purchased from Chemicon International, Inc. (Temecula, CA).
Horseradish peroxidase-conjugated rat affinity-purified antibodies to
mouse IgM, mouse IgG, and rabbit IgG were purchased from
Zymed Laboratories Inc. (San Francisco, CA). Rat anti-mouse IgM antibodies were purchased from Cappel (West Chester, PA). Protein
G-Sepharose resins and ECL reagents were purchased from Amersham
Biosciences. Polyvinylidene difluoride (PVDF) membrane (Immobilon P)
was a product of Millipore (Bedford, MA). Prestained molecular weight
marker was purchased from Bio-Rad (Hercules, CA). BCA protein
assay kit was purchased from Pierce.
1,2-Dimano-3,4-methylenedioxybenzene (DMB) was purchased from
Dojindo (Kumamoto, Japan). Peptide:N-glycanase (PNGase
F) was purchased from Takara (Kyoto, Japan).
Chemical Analysis--
Sialic acids were quantitated by the
fluorometric analysis using
-keto acid-specific reagent, DMB (32).
The oligo/polysialic acids were determined by the fluorometric
C7/C9 analysis (28) and by the mild acid
hydrolysis/fluorometric analysis as described previously (29).
Concentration of proteins was determined by the BCA protein assay kit
or by the measurement of absorbance at 280 nm.
SDS-PAGE and Immunostaining--
Pig embryonic brain homogenates
were prepared as described previously (12). Samples treated with or
without PNGase F (1.0 milliunit) at 37 °C for 20 h were
dissolved in Laemmli buffer with or without 5% mercaptoethanol and
placed at 60 °C for 20 min. The samples were then electrophoresed on
7.5% polyacrylamide gels and visualized by silver staining or
electroblotted on the PVDF membrane using a semidry blotting apparatus.
The PVDF membrane was blocked with 10 mM sodium phosphate
buffer (pH 7.2), 0.15 M NaCl (PBS) containing 0.05% Tween
20, and 1% bovine serum albumin at 37 °C for 1 h. After
treatment with or without Endo-N (0.009-9.0 unit/ml) at 37 °C for
17 h or 0.1 N NaOH at 37 °C for 20 h, the membranes were incubated with the primary antibody mAb 12E3 (0.36 µg/ml), mAb 735 (1.0 µg/ml), mAb OL28 antibody (35.2 µg/ml), mAb S2
566 antibody (1.0 µg/ml), anti-NCAM (2.0 µg/ml), or anti-CD36 (0.40 µg/ml) 4 °C for 16 h. As the secondary antibody,
peroxidase-conjugated anti-mouse IgM (1:5000 dilution), anti-mouse IgG
(1:5000 dilution), and anti-rabbit IgG (1:5000 dilution) were used at
37 °C for 45 min and the color development was carried out as
described previously (12).
Immunoprecipitation--
Milk (3.3 mg/ml as BSA, 100 µl) was
pretreated with 100 µl of 1:1 solution of protein G-Sepharose coupled
with rat anti-mouse IgM antibodies at 4 °C for 1 h, and resins
were removed. The pretreated milk was incubated with protein
G-Sepharose (100 µl) coupled with mAb 12E3 via rat anti-mouse IgM
antibodies at 4 °C for 16 h. The resins were washed with four
times with PBS containing 0.05% Tween 20 and subjected to
SDS-PAGE/Western blotting. For immunoprecipitation with anti-CD36, 100 µl of milk that had been pretreated with protein G-Sepharose as
described above was incubated with 100 µl of protein G-Sepharose
coupled with anti-CD36 at 4 °C for 16 h. The immunoprecipitate was immunoblotted using anti-CD36 and mAb 12E3.
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RESULTS |
Detection of PolySia in Human Milk by Chemical Methods--
We
previously demonstrated that several glycoproteins in brain, adipose
tissue, and blood have the diSia and oligoSia structures (12, 22, 23,
25-27). Further attempts to search for the di/oligo/polySia structures
on glycoproteins in other cells and tissues suggested that mouse
mammary epithelial cells contained such structures, because high
amounts of internal Neu5Ac (
8Neu5Ac) residues were detected using
C7/C9 analysis as well as by immunochemical
methods (30). Thus, we began to test whether milk contains the polySia structure. We first performed mild acid hydrolysis/fluorometric analysis of human milk. Milk was treated with mild acid, and released oligo/polySia residues were labeled with DMB followed by high performance liquid chromatography on a Resource Q-anion exchange column. When colominic acid, a homo-oligomer and polymer of
2
8-linked Neu5Ac, was subjected to these procedures, Neu5Ac
dimers of up to octadecamers were successfully separated (Fig.
1a). With milk, peaks of dimer
to at least octadecamer were also detected (Fig. 1b). These
results indicate that human milk contains a polySia structure with DP
ranging from 2 to at least 18.

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Fig. 1.
Resource Q anion exchange chromatography of
2 8-linked oligo/polyNeu5Ac-DMB released by mild acid
hydrolysis. 2 8-Linked oligo/polyNeu5Ac was
labeled with DMB and applied to a Resource Q anion exchange column (1 ml, Cl form). The column was eluted with 20 mM Tris-HCl (pH 8.0) with a gradient from 0-0.3
M NaCl as indicated by a line. The elution was
monitored by a fluorescence detector (set at wavelength of 373-nm
excitation and 448-nm emission). a, colominic acid (1 µg).
b, human milk sample (1,650 µg as BSA) was treated with
0.01 N trifluoroacetic acid at 50 °C for 1 h and
subjected to DMB-labeling high performance liquid chromatography
analysis as described under "Experimental Procedures."
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Detection of PolySia in the 85-kDa Glycoprotein in Human
Milk--
The presence of the polySia structure in human milk was also
detected using Western blotting with mAb 12E3, which specifically recognizes
2
8-linked oligo/polyNeu5Ac with DP
5. As shown in Fig. 2, b and c
(lane 1), the 85-kDa glycoprotein was reactive with mAb
12E3. These results indicate that the 85-kDa gp contains
2
8-linked polyNeu5Ac structure. The 85-kDa gp was not
reactive with anti-NCAM antibodies, whereas a smear band of a high
molecular mass region of embryonic pig brain homogenate was detected
with both mAb 12E3 and the anti-NCAM antibodies (lane 2).
This finding indicates that the 85-kDa gp is different from
polySia-containing NCAM.

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Fig. 2.
SDS-PAGE/silver stain and Western blot
analysis of human milk. 16.5 µg (as BSA) of human milk
(lane 1) and 10 µg of pig embryonic brain homogenates (as
BSA) (lane 2) were separated on SDS-polyacrylamide gel
(7.5%) under the reducing condition and subjected to the silver
staining (a) or blotted on the PVDF membrane. The membrane
was incubated with (1st Ab. +) or without (1st Ab. ) mAb 12E3 (0.72 µg/ml) (b) or anti-NCAM antibodies (2.0 µg/ml)
(c) and visualized.
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Identification of the 85-kDa gp as CD36--
Many studies have
been done on human milk components (33). Based on its molecular mass,
we hypothesized that the 85-kDa gp might be CD36. As described
previously (34), the human milk CD36 was detected at 85 kDa as one of
the major components on SDS-PAGE/silver-stained gels (Fig.
2a). To determine whether the 85-kDa gp is CD36, CD36 was
immunopurified using anti-CD36 and analyzed with mAb 12E3. The
immunopurified CD36 migrated at 85 kDa (Fig.
3a, Blot:
anti-CD36) and was immunostained with mAb 12E3 (Fig.
3a, Blot: 12E3). Similarly, the
immunoprecipitate of milk with mAb 12E3 was immunostained at 85 kDa
with anti-CD36 (Fig. 3b). These results indicate that the
85-kDa gp is a CD36 containing the polySia structure. To address the
question of whether all milk CD36 molecules are polysialylated, we
performed quantitative analysis of the CD36 that contains the
2
8-linked polySia. The intensity of the anti-CD36 stain of
the immunoprecipitate of human milk with mAb 12E3 was densitometrically
compared with that of the unprecipitated CD36 and indicated that 4.6%
of total CD36 molecules were polysialylated.

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Fig. 3.
Immunodetection of the 12E3 epitope in the
immunopurified CD36 from human milk. a, CD36 molecule
was immunopurified from human milk using rabbit anti-CD36 antibodies.
Immunopurified CD36 (IP: anti-CD36) was subjected
to the SDS-PAGE and blotted on the PVDF membrane. The membrane was then
immunodetected with anti-CD36 antibodies (Blot:
anti-CD36) or mAb 12E3 (Blot: 12E3)
and visualized as described. b, oligo/polysialylated
molecules were immunopurified from human milk using mAb 12E3, which
specifically recognizes (Neu5Ac)n, n 5. Immunopurified oligo/polysialylated molecule (IP:
12E3) was subjected to the SDS-PAGE and blotted on the PVDF
membrane. The membrane was then immunodetected with anti-CD36
antibodies (Blot: anti-CD36) or mAb 12E3
(Blot: 12E3) and visualized as described.
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Characterization of the PolySia Chain on the Human Milk
CD36--
To confirm that milk CD36 contains
2
8-linked
polySia, we used Endo-N as a specific probe for detection of
2
8-linked polySia. Human milk was subjected to SDS-PAGE
followed by electroblotting on PVDF membranes. The membranes were
treated or untreated with 0.009-9 milliunit/ml Endo-N and then
immunodetected with mAb 12E3. As shown in Fig.
4a
(Blot:12E3), immunostain with mAb 12E3
disappeared with increasing amounts of Endo-N. At 0.9 milliunit or more
of Endo-N, the mAb 12E3 immunostain was clearly lost, whereas the anti-CD36 immunostain remained unchanged. These results indicate that
milk CD36 contains the
2
8-linked polySia with DP
5 and
Endo-N digestion has no effect on the antigenicity of CD36 recognized
by anti-CD36.

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Fig. 4.
Western blot analysis of human milk with
Endo-N and various anti-di/oligo/polysialic acid antibodies.
a, human milk (17 µg/lane) was subjected to the SDS-PAGE
and electroblotted on the PVDF membrane. The membranes were then
blocked with PBS containing 1% BSA and digested with 0.009-9
milliunits of Endo-N or without ( ) Endo-N at 37 °C for 12 h.
The Endo-N-treated or untreated membranes were then incubated with mAb
12E3 (Blot: 12E3) or with anti-CD36 antibodies
(Blot: anti-CD36) and immunodetected using
peroxidase-conjugated anti-mouse IgM (1:5000 dilution) for mAb 12E3 and
anti-rabbit IgG antibodies (1:3000 dilution) and ECL reagents.
b, glycoproteins were separated with 7.5%
SDS-polyacrylamide gel and electroblotted on PVDF membrane. The
membranes were immunostained using anti-polySia antibody (mAb
735) (0.06 µg/ml), anti-oligo + polySia antibody (mAb
12E3) (0.72 µg/ml), anti-oligo + polySia antibody (mAb
OL28) (0.4 µg/ml), anti-disialylGal antibody (mAb
S2-566) (1.0 µg/ml), and anti-CD36 antibodies (0.4 µg/ml) as described under "Experimental Procedures."
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We previously demonstrated that a group of anti-di/oligo/polySia
antibodies with defined immunospecificities is useful for determination
of the chain length of polySia (12). We used four antibodies, mAb 735 (DP
11), mAb 12E3 (DP
5), mAb OL28 (DP
4), and mAb S2-566
(specific for the diSia structure
Neu5Ac
2
8Neu5Ac
2
3Gal) for immunodetection. As shown
in Fig. 4b, human milk CD36 was immunostained by all of
these antibodies. The fact that milk CD36 was recognized by mAb735
suggests that the DP of the polySia chain is 11 or greater. This is
consistent with the results of high performance liquid chromatography
analysis of mild acid hydrolysate of human milk (Fig. 1b).
The milk CD36 was also reactive with mAb S2-566, suggesting that CD36
contains the diSia as well as a polySia structure.
To determine whether polySia is present on the N-linked or
O-linked glycan(s), we performed PNGase F digestion and
alkaline treatment (0.1 N NaOH at 37 °C for 20 h).
The PNGase F digestion resulted in the reduction of the molecular mass
of milk CD36 from 85 to 82 kDa without losing mAb 12E3 immunostain on
milk CD36 (Fig. 5a). On the
other hand, the mAb 12E3 immunostaining of milk CD36 was lost after
alkaline treatment. The alkaline treatment had no effect on anti-CD36
antibody immunostaining. These findings indicate that the polySia
epitope is present on O-linked glycan(s) in milk CD36.

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Fig. 5.
Western blot analysis of human milk after
treatment with PNGase F or 0.1 N NaOH. a,
41 µg (as BSA) of human milk was treated with or without PNGase F
(1.0 milliunits) at 37 °C for 20 h. PNGase F-untreated human
milk (lane 1), PNGase F-treated human milk (lane
2), and 20 µg (as BSA) of embryonic pig brain homogenates
(lane 3) were subjected to the 7.5% SDS-polyacrylamide gel.
After being blotted on the PVDF membrane, glycoproteins were
immunodetected with mAb 12E3 (0.72 µg/ml) (Blot:
12E3) or anti-CD36 antibodies (Blot:
anti-CD36 antibodies) (0.40 µg/ml). b, 41 µg
(as BSA) of human milk (lane 1) and 20 µg (as BSA) of
embryonic pig brain homogenates (lane 3) were subjected to
the 7.5% SDS-polyacrylamide gel and electroblotted on the PVDF
membrane. The membrane was treated with 0.1 N NaOH at
37 °C for 20 h for -elimination (to remove
O-linked glycan chains) and detected using mAb 12E3
(Blot: 12E3) (0.72 µg/ml) or anti-CD36
antibodies (Blot: anti-CD36 antibodies) (0.40 µg/ml).
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PolySia Is Present in Milk CD36 but Not in Platelet CD36 in
Human--
CD36 is present in platelets as well as in milk. To
determine whether the polySia on CD36 is common between milk and
platelets, platelet CD36 was prepared and tested for the presence of
polySia. The platelet CD36 migrated at 88 kDa on
SDS-PAGE/immunostaining with anti-CD36, whereas milk CD36 migrated at
85 kDa (Fig. 6, lanes 1 and 2)
as reported previously (34). Platelet CD36 was not immunostained with
mAb 12E3 in contrast with milk CD36 (Figs. 3b and 6). This
finding indicates that polysialylation of CD36 is specific to
milk.

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Fig. 6.
Western blot analysis of human platelet CD36
with mAb 12E3. 16.5 µg (as BSA) of human milk (lane
1) and 16.4 µg (as BSA) of human platelet homogenates
(lane 2) were subjected to the SDS-PAGE/Western blot
analysis using anti-oligo + polySia antibody (mAb 12E3)
(0.72 µg/ml) and anti-CD36 antibodies (0.40 µg/ml). Human milk CD36
is 85-kDa glycoprotein, and human platelet CD36 is a 88-kDa
glycoprotein.
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The Presence of PolySia in Mouse Milk CD36--
To determine
whether the polySia on milk CD36 is common among mammals, we analyzed
mouse milk using Western blot with mAb 12E3. Mouse milk was reactive
with mAb 12E3 at an expected molecular mass (82 kDa) of mouse milk CD36
(35) (Fig. 7). Immunostaining of these
CD36 with mAb 12E3 was completely lost following Endo-N digestion,
whereas the anti-CD36 immunostaining remained unchanged (Fig. 7,
Blot: anti-CD36). These results indicate that the
polySia on milk CD36 is common between human and mouse.

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Fig. 7.
Western blot analysis of mouse milk before
and after digestion with Endo-N. Human and mouse milk (17 µg/lane) were subjected to the SDS-PAGE and electroblotted on the
PVDF membrane. The membranes were then blocked with PBS containing 1%
BSA and digested with 9.0 milliunits of Endo-N or without ( ) Endo-N
at 37 °C for 12 h. The Endo-N-treated or untreated membranes
were then incubated with mAb 12E3 (Blot: 12E3) or
with anti-CD36 antibodies (Blot: anti-CD36) and
immunodetected using peroxidase-conjugated anti-mouse IgM (1:5000
dilution) for mAb 12E3 and anti-rabbit IgG antibodies (1:3000 dilution)
and ECL reagents.
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Developmental Expression of the PolySia on CD36 during
Lactation--
The amounts of the polySia epitope and milk CD36 in
colostrum within a week after parturition and milk within 1, 3, and 6 months after parturition were analyzed using Western blot with mAb 12E3
and anti-CD36. The intensity of the bands immunostained with mAb 12E3
and anti-CD36 was quantified densitometrically. The results are
summarized in Table I. The quantity of
milk CD36 was highest in colostrum. During lactation, the quantity of
CD36 in milk reached a minimum of 1 month after parturition and then increased and became constant at 3 and 6 months after parturition. With
respect to the polySia on milk CD36, the quantity of the polySia on
milk CD36 1 month following parturition was largely constant and
slightly larger than that of the colostrum CD36. The proportions of
polySia to CD36 reached a maximum in milk at 1 month after parturition
and gradually decreased as the lactation stage progressed. The
proportion in the colostrum was lowest during lactation. These results
suggest that polysialylation of milk CD36 takes place maximally at 1 month after parturition.
 |
DISCUSSION |
To our knowledge, the only two glycoproteins that contain polySia
in mammals are NCAM (4, 9, 10) and the sodium channel
-subunit (11).
The polySia NCAM occurs in embryonic brain, hippocampus, and olfactory
bulb in adult brain, kidney, heart, muscle (4, 9), and some tumors such
as neuroblastoma, nephroblastoma, medulloblastoma, pheochromocytomas,
medullary carcinomas of thyroid, small cell lung carcinoma, and
pituitary adenoma (4, 21). Functions of polySia NCAM in neural tissues
are well documented (9). PolySia residues inhibit cell-cell and
cell-matrix interactions and regulate neural development. PolySia
epitopes on tumor cells are a marker of tumor stages (36) and are
considered to be involved in metastasis (20, 21). PolySia is also
present in sodium channels. The eel electroplax sodium channel was the
first such example (37), and later, it was reported in the
-subunit
of voltage-sensitive sodium channel in adult rat brain (11). However, the function of polySia on these sodium channels has not yet been elucidated. In this study, we discovered milk CD36 as a third member of
polySia-containing glycoproteins in mammals using highly sensitive
methods that we developed including two chemical analyses, fluorometric
C7/C9 analysis (28) and mild acid
hydrolysis/fluorometric analysis (29), and immunochemical methods using
Endo-N and a group of antibodies with defined immunospecificity (12).
Human milk CD36 contains a polySia chain with a DP of at least 18. The polySia structure is attached to O-linked glycan chain(s)
but not to N-linked glycan chains. This feature is in
contrast with the NCAM-containing polySia on N-linked glycan
chains (4).
In 1996, Colley and colleagues (38) reported the presence of unique
glycoproteins containing polySia structures on O-linked glycan chains in MCF7 breast cancer cells and RBL basophilic
leukemia cells. These glycoproteins were different from NCAM or the
sodium channel
-subunit, although they were not identified at that
time (38). The polySia-containing glycoproteins in MCF7 cells have a
molecular mass of 180-260 kDa, whereas that of human milk CD36 is 85 kDa. Therefore, these glycoproteins are different from CD36 and remain
to be elucidated. Milk CD36 is considered to be synthesized in and
secreted from the mammary gland (39). Thus, it is suggested that
sialyltransferases responsible for the synthesis of polySia on
O-linked glycan chains are present in the mammary gland.
Because MCF7 cells are derived from human mammary glands,
sialyltransferases involved in the synthesis of the polySia on
180-260-kDa glycoproteins might be the same ones used for synthesis of
the polySia CD36 in the mammary gland. Two enzymes, PST (ST8Sia IV) and
STX (ST8Sia II), are now known to be responsible for the synthesis of
polySia on N-linked glycan chains of NCAM (40, 41). It
remains to be elucidated whether these two enzymes are involved in the
synthesis of polySia on the O-linked glycan chains of CD36
and the 180-260-kDa glycoproteins. In this regard, we recently
reported in a preliminary form that these two enzymes are also involved
in the synthesis of polySia on O-linked glycan chains based
on studies with fish egg polysialoglycoprotein (42). Elucidation of a
biosynthetic mechanism for polySia formation on O-liked
glycan chains in mammalian cells is under way in our laboratory.
Another interesting feature of the glycan structures of milk CD36 is
the presence of the diSia epitope,
Neu5Ac
2
8Neu5Ac
2
3Gal sequence, on
O-linked glycan chains. Thus, milk CD36 is a new member of
the diSia-containing glycoprotein family that was recently established
by our group (12, 22, 23, 25-27). This family includes adiponectin
(25) from adipose tissue and CD166 (SC1) from brain and T lymphocytes
(27). The polySia epitope was not detected in these two glycoproteins.
We do not know at present whether the diSia epitope in milk CD36 is an
intermediate form of polySia chain or is linked to a site independent
of the polySia-bearing site in milk CD36. Structural determination of the O-linked glycan chains of milk CD36 with and without the
polySia epitope will be necessary.
The presence of polySia on CD36 appears to be limited to milk. CD36 is
a member of the B class of the scavenger receptor superfamily and occurs in milk and blood cells including platelets and some types
of macrophages (43). CD36 is likely to have diverse functions. First,
platelet CD36 acts as a receptor for collagen and thrombospondin leading to platelet activation (44-46) and
thrombospondin-mediated inhibition of angiogenesis (47). Second, CD36
in hemopoietic cells acts as a scavenger receptor through binding to
apoptotic cells and cell fragments via anionic phospholipids (48). CD36 in blood can also bind oxidized low density lipoproteins (49) and
advanced glycation end products (50) to eliminate them from systemic
circulation. Third, CD36 acts as a constitutive fatty acid transporter
of long chain fatty acids (51). On the other hand, the function of milk
CD36 has not yet been elucidated. CD36 is integrated in milk fat
globule membrane, a layer of milk droplets secreted from mammary
epithelial cells (39). In this study, we demonstrated that milk CD36,
but not platelet CD36, is polysialylated. Milk CD36 of not only human
but also mouse is modified by polySia. In human milk, 4.6% CD36 is
polysialylated. The level of the polySia in milk CD36 changes during
lactation and is maximized 1 month after parturition. The ubiquitous
presence in mammals and the lactation stage-dependent
expression of the polySia CD36 in milk suggest that polysialylation of
milk CD36 is important for the development of mammalian neonates.
As a milk component, protection and nutrition of neonates are important
aspects to be considered for the polySia of milk CD36. As a protective
material, the polySia of milk CD36 can be a natural trap for various
pathogenic viruses and bacteria to prevent their invasion into
neonates, because sialic acids are often major receptors for such
pathogens (52). Glycosylation states of CD36 affect the binding
properties of CD36 to oxidized low density lipoprotein (53). The
altered binding to the oxidized fat materials by the presence of
polySia might prevent such harmful materials from being incorporated
into neonates. Another interesting aspect of polySia function in milk
is nutrition. Human milk is a rich source of sialic acid (54), although
the significance and metabolic fate of the sialic acids as a nutrient
are currently unknown. Morgan and Winick (55) reported that sialic acid
exogenously administered by intraperitoneal injection increases
production of gangliosides in brain and improves the learning of both
well nourished and malnourished rat pups. Carlson and House (24) reported that both intraperitoneally and orally administered sialic acids increase gangliosides in young rat brain. Thus, the supply of
sialic acids to neonates is important for development of the neural
system. For these purposes, in addition to de novo synthesis of sialic acids, neonates might incorporate sialic acids from milk.
Monomeric forms of sialic acids and polySia might both be good sources
of sialic acid.