Polysialic Acid in Human Milk

CD36 IS A NEW MEMBER OF MAMMALIAN POLYSIALIC ACID-CONTAINING GLYCOPROTEIN*

Uichiro YabeDagger , Chihiro SatoDagger , Tsukasa Matsuda, and Ken KitajimaDagger §

From the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, the Dagger  Department of Animal Sciences, Division of Organogenesis, Nagoya University Bioscience Center, Nagoya 464-8601, Japan, and the § Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan

Received for publication, January 15, 2003, and in revised form, February 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The neural cell adhesion molecule and the voltage-sensitive sodium channel alpha -subunit are the only two molecules in mammals known to be modified by alpha -2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide:N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha right-arrow 5O-glycolyl, alpha right-arrow 8, alpha right-arrow 9, and alpha right-arrow 8/9) (4). alpha right-arrow 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 alpha -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Neu5Acalpha right-arrow 8 Neu5Acalpha 2 right-arrow 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 alpha -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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (right-arrow8Neu5Ac) 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 alpha 2 right-arrow 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 alpha right-arrow 8-linked oligo/polyNeu5Ac-DMB released by mild acid hydrolysis. alpha right-arrow 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."

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 alpha right-arrow 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 alpha right-arrow 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.

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 alpha right-arrow 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.

Characterization of the PolySia Chain on the Human Milk CD36-- To confirm that milk CD36 contains alpha right-arrow 8-linked polySia, we used Endo-N as a specific probe for detection of alpha right-arrow 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 alpha right-arrow 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."

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 Neu5Acalpha 2 right-arrow 8Neu5Acalpha right-arrow 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 beta -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).

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.

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.

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.


                              
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Table I
Developmental expression of the polySia on the milk CD36 during lactation


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To our knowledge, the only two glycoproteins that contain polySia in mammals are NCAM (4, 9, 10) and the sodium channel alpha -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 alpha -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 alpha -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, Neu5Acalpha right-arrow 8Neu5Acalpha 2 right-arrow 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.

    FOOTNOTES

* This work was supported in part by grants-in-aid for the 21st Century COE Program (to K. K.), for CREST of Japan Science and Technology Corporation (to K. K.), and for Young Scientists (B) 14780471 (to C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Animal Sciences, Division of Organogenesis, Nagoya University Bioscience Center, Nagoya 464-8601, Japan. Tel.: 81-52-789-4297; Fax: 81-52-789-4297; E-mail: kitajima@agr.nagoya-u.ac.jp.

Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M300458200

    ABBREVIATIONS

The abbreviations used are: Sia, sialic acids; Neu5AC, N-acetylneuraminic acid; polySia, polysialic acid; DP, degree of polymerization; BSA, bovine serum albumin; diSia, disialic acid; NCAM, neural cell adhesion molecules; Endo-N, Endo-N-acylneuraminidase; mAb, monoclonal antibody; DMB, 1,2-diamino-4,5-methylenedioxybenzene; PNGase F, peptide:N-glycanase; oligoSia, oligosialic acid; PBS, phosphate-buffered saline; gp, glycoprotein; PVDF, polyvinylidene difluoride; ST8Sia, alpha -2,8-sialyltransferase; BSA, bovine serum albumin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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