Purification and cDNA Cloning of Porcine Brain GDP-L-Fuc:N-Acetyl-β-D-Glucosaminide α1→6Fucosyltransferase

GDP-L-Fuc:N-acetyl-β-D-glucosaminide α1→6fucosyltransferase (α1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-2)Manβ1-4GlcNAcβ1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440,000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The α1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. α1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that α1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.

It has been reported that the structures of glycopeptides change during the development and differentiation of embryos (1)(2)(3)(4). Detailed analysis of specific antigens on the surface of various carcinoma cells revealed that carcinoma-specific sugar chains are expressed on the cell surface. A well documented phenotypic alteration of these specific sugar chains is the increase in the molecular weight of cell surface complex type N-linked glycan in transformed cells. This change has been observed regardless of the nature of the transforming agent: oncogenic viruses (5)(6)(7)(8)(9), chemical mutagens (10 -11), or DNA from unrelated tumor cells (12)(13)(14). This phenomenon was thought to reflect the deviation of carcinoma cells from the ordinary differentiation processes. ␣-Fucose residue attached to asparagine-linked GlcNAc also have some relationship with carcinogenesis. A difference in the binding pattern of serum ␣-fetoprotein with lentil lectin between hepatocellular carcinomas and benign liver diseases has been reported (15)(16)(17). Analyses of the carbohydrate structure of ␣-fetoprotein from hepatocellular carcinoma cell lines have indicated that almost all of the carbohydrates of ␣-fetoprotein are ␣1-6-fucosylated (18). ␣-Fetoprotein produced by germ cell tumors, such as yolk sac tumors, is also highly fucosylated (19). The activity of ␣1-6FucT 1 was higher in hepatocellular carcinoma tissue than in non-tumor tissue (20) and was induced by the transfection of the ras protooncogene into 3T3 fibroblast cells (21).
Schachter et al. (22,23) first characterized ␣1-6FucT in porcine liver using a partially purified enzyme extract. The special release of ␣1-6FucT from platelets during blood clotting has been reported (24,25), alteration of fucosylation has been reported in cystic fibrosis glycoproteins from different sources, and ␣1-6FucT from human fibroblasts of cystic fibrosis patients has been purified and characterized (26,27). But little is known about this enzyme, and the ␣1-6FucT gene has not yet been cloned. In this study we purified a novel ␣1-6FucT, which is a different enzyme from the ␣1-6FucTs previously reported, especially in terms of the pH optimum and molecular weight. The cDNA sequence of this enzyme was also determined for the first time. A highly specific assay method involving a fluorescent reagent, 4-(2-pyridylamino)butylamine (PABA), made it possible to purify the enzyme (28). EXPERIMENTAL PROCEDURES 2-Amino pyridine was obtained from Wako Pure Chemicals. ␤-Galactosidase (Aspergillus sp.) was obtained from Toyobo Co. TSK-gel ODS-80TM and Amide-80 columns were purchased from Tosoh. Bovine ␥-globulin and Pronase (Streptomyces griseus) were purchased from Sigma and Seikagaku Kogyo Co., respectively.

Preparation of Substrate for ␣1-6FucT
Fluorescence-labeled oligosaccharides were obtained from the corresponding oligosaccharides as described previously (28). Briefly, a glycopeptide derived from bovine ␥-globulin on digestion with Pronase was coupled with PABA using water-soluble carbodimide. Further purification was performed by HPLC on a TSK-gel ODS-80TM column (4.6 ϫ 150 mm) that had been equilibrated with 20 mM acetate buffer, pH 4.0, containing 0.1% butanol, at a flow rate of 1 ml/min isocratically. The eluted pyridylamino sugars were detected with a fluorescence spectrophotometer, the excitation and emission wavelengths being 320 and 400 nm, respectively. * This study was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science and Culture of Japan, the Uehara Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D86723.

␣1-6FucT Assay
The enzyme activity was determined as described (28). In brief, the standard incubation mixture for the ␣1-6FucT assay contained the following components, in a final volume of 50 l: 200 mM MES-NaOH buffer, pH 7.0, 1% Triton X-100, 50 M acceptor (GnGn-Asn-PABA), and 500 M donor (GDP-Fuc) substrates. After the mixture had been incubated at 37°C for 2 h, the reaction was stopped by heating at 100°C for 1 min. The sample was centrifuged at 15,000 ϫ g for 10 min, and then 10 l of the supernatant was used for analyses. The product was separated by HPLC on a TSK-gel ODS-80TM column (4.6 ϫ 150 mm). Elution was performed at 55°C with 20 mM acetate buffer, pH 4.0, containing 0.1% butanol in an isocratic way. Fluorescence of the column eluate was detected with a fluorescence spectrophotometer (model RF 535; Shimadzu, Japan), the excitation and emission wavelengths being 320 and 400 nm, respectively. The amount of product was estimated from the fluorescence intensity. The specific activity of the enzyme was expressed as pmol of fucose transferred/h/mg of protein. Protein was determined with a Bio-Rad protein assay kit using bovine serum albumin as a standard.

Preparation of GDP-Hexanolamine
GDP-hexanolamine was synthesized by the method of Nunez, et al. (29) with a slight modification. Briefly, GMP was activated with morphoridate and then coupled to 6-aminohexylphosphate. The end product was completely purified on a TSK gel ODS TM80 column (4.6 ϫ 150 mm). The structure of the GDP-hexanolamine column was confirmed by 1 H NMR analyses (data not shown).
Step 2-After centrifugation at 900 ϫ g for 10 min, the resultant supernatant was centrifuged at 105,000 ϫ g for 1 h. The pellet was resuspended in a 1000-ml solution of 20 mM phosphate-KOH, pH 7.0, 50 mM KCl, 5 mM EDTA, and 0.5% Triton X-100 buffer and then extracted by gentle stirring for 7 days using a magnetic stirrer, followed by centrifugation at 105,000 ϫ g for 1 h. The supernatant was collected and concentrated to 75 ml using a YM 30 membrane (Amicon).
Step 3-The above extract was applied to a column (16 ϫ 5 cm; Pharmacia HR 16/10) of GnGn-bi-Asn-Sepharose 4B, which had been equilibrated with the 20 mM phosphate-KOH, pH 7.0, 50 mM KCl, 5 mM EDTA, and 0.5% Triton X-100 buffer. The column was washed with the same buffer and then eluted with the washing buffer containing 1 M KCl.

Isoelectric Focusing and SDS-PAGE
Isoelectric focusing gel electrophoresis was carried out on a pI 3-9 gradient gel (Pharmacia) according to the manufacturer's instructions using 1 g of the purified enzyme. At the same time, another 1 g of the purified enzyme was applied to another lane of the same gel. After electrophoresis, one lane was cut into pieces, and each slice was sonicated and extracted with the assay mixture. Then ␣1-6FucT activity was measured. At the same time, the remaining lane was stained with silver utilizing Sil-Best Stain (Nacalai Tesque) according to the manufacturer's instructions. SDS-PAGE was performed by the method described by Laemmli (30) in the presence or absence of 2-mercaptoethanol. The gels were stained with Coomassie Brilliant Blue G-250 and silver.

Determination of Partial Amino Acid Sequences of ␣1-6FucT
To determine the N-terminal amino acid sequences, the material corresponding to the 54-kDa band was excised from a polyvinylidene difluoride membrane (Millipore) and then sequenced with an Applied Biosystem 473A Protein Sequencer. To determine internal amino acid sequences, 13 g of the purified protein transferred onto a polyvinylidene difluoride membrane was digested with 1 g of lysyl endopeptidase in 100 mM Tris and 5% acetonitrile, pH 8.2, at 37°C for 12 h. The resultant peptides were separated by reverse phase HPLC on a Vydac C-18 column (250 ϫ 2.1 mm), using a 0 -60% acetonitrile gradient in 0.05% trifluoroacetic acid. Five peptides were obtained and applied to a Polybrene-coated trifluoroacetic acid-activated precycled glass fiber filter for amino acid sequencing and were analyzed with an Appled Biosystems 473A protein sequencer.

Construction of the cDNA Encoding ␣1-6FucT cDNA
Total RNA was isolated from a porcine brain according to general methods. Poly(A) ϩ was further purified on an oligo(dT)-cellulose (Pharmacia) column. The first strand cDNA was synthesized with a cDNA synthesis kit (Life Technologies, Inc.), using primer A11, according to the manufacturer's manual (see Fig. 8).

Polymerase Chain Reaction (PCR)
Four oligonucleotides were synthesized for use as primers in PCR (see Fig. 7). PCR was carried out in 50-l reaction mixtures containing first strand cDNA corresponding to 1 g of poly(A) ϩ RNA, 50 pmol of a pair of degenerate oligonucleotides, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 0.001% gelatin, and 200 M dNTP. After a hot start at 98°C, 25 cycles (94°C for 1 min, 55°C for 2 min, 72°C for 3 min) of PCR were performed using 2.5 units of Thermus aquaticus (Taq) polymerase. The PCR products were subcloned into a pT7BlueT-Vector (Novagen).

Isolation and DNA Sequencing of Porcine ␣1-6FucT cDNA
A porcine brain gt11 cDNA library (Clonetech) was screened by plaque hybridization utilizing the [␣-32 P]dCTP (3,000 Ci/mmol; Amersham) of a labeled reverse transcription-PCR product, S-A12 (see Fig.  8). Five positive plaques were obtained from 4 ϫ 10 5 plaques in the first screening. The screening was repeated 3 times, and then the inserts of the five isolated clones were amplified using forward (GGTGGCGAC-GACTCCTGGAGCCCG) and reversal (CGTGTATTCCTCAATGGGAT-GGAA) primers by means of PCR reaction, and the DNA sequence was directly determined with a DNA Sequencing Kit of Dye Terminator Cycle Sequencing Ready Reaction (Perkin-Elmer) using forward and reversal primers as sequencing primers. Based on the determined insert cDNA sequence, sequencing primers were synthesized, and the space between each sequencing primer was 200 nucleotides. Finally the full length of the insert cDNA sequence was determined.

Transient Expression of ␣1-6FucT in COS-1 Cells
The coding region of cDNA of ␣1-6FucT was amplified using forward (ggaattccGAGTTGAAAGTCTGAAAATGCGG) and reversal (tccccc-gggggaCTTTCTCATCTGTCCGTC) primers by means of PCR reaction. The obtained PCR product was double digested by SmaI and EcoRI and subcloned into calf intestinal alkaline phosphatase-treated pSVK3, which was previously double digested by SmaI and EcoRI. pSVK3 was then transiently transfected into COS-1 cells by electroporation (Bio-Rad).

Northern Blotting Analyses of ␣1-6FucT mRNA in Rat Brain
Total RNA was prepared from rat cerebrum and cerebellum according to general methods. 20 g of total RNA was chromatographed on a 0.8% agarose gel containing 2.2 M formaldehyde. The size-fractionated RNA was transferred to a -Probe membrane (Bio-Rad) by capillary action. After hybridization with 32 P-labeled S-A12 fragment (see Fig. 9) at 42°C, the membrane was washed at 55°C in 2 ϫ SSC (1 ϫ SSC: 15 mM sodium citrate and 150 mM NaCl, pH 7.0) containing 0.1% SDS. The Kodak x-ray film was exposed for 3 days at Ϫ80°C.

Purification and Column Chromatography of ␣1-6FucT
Steps 1 and 2-A porcine brain was chosen as a source of the enzyme for purification based on the fact that porcine and rat brains are the most abundant sources of the enzyme (Table I).
Step 3-The first chromatographic step of the purification involved fractionation on a GnGn-bi-Asn-Sepharose 4B column. GnGn-bi-Asn-Sepharose 4B chromatography resulted in a 61% yield and 2,800-fold purification (Table II). The majority of total protein loaded was eluted in the flow-through fraction of a column, as shown in Fig. 1. Although most glycosyltransferases and lectins were not retained by the column, porcine brain ␣1-6FucT is fully active in EDTA, as discussed below. From the beginning, enzyme activity began to be eluted gradually from the column, suggesting that the ␣1-6FucT exhibits heterogeneity. This is likely to be the reason why a 60% recovery of the total activity loaded was achieved when the column was eluted with KCl. Step 4 -After the buffer had been changed using an Amicon YM30, the final purification step was accomplished on a synthetic GDP-hexanolamine ligand column. Chromatography on an immobilized GDP-hexanolamine column resulted in an additional 155-fold purification with a 19.7% yield compared with the previous step. The elution profile from this column clearly showed that the majority of the contaminating protein was not bound to the column and that the enzyme was eluted from this column with GDP.
Starting with frozen porcine brain (100 g), ␣1-6FucT was purified 440,000-fold with a total yield of 12%, as summarized in Table II.

Purity and Enzymatic Properties of ␣1-6FucT
In order to assess the level of purification of ␣1-6FucT, fractions eluted from a GDP-hexanolamine column were subjected to SDS-PAGE. A photograph of this gel, which was stained with Coomassie Brilliant Blue G-250 is shown in Fig. 2. The most purified sample in lanes 28 and 29 gave one major band corresponding to an apparent mass of 58 kDa (indicated by an arrow on the right).
The enzymatic profiles of ␣1-6FucT of porcine brain were obtained utilizing the purified protein. The purified enzyme was incubated by the enzyme assay method, and an aliquot was subjected to HPLC. A typical elution pattern is shown in Fig. 3. The substrate and product were eluted at 12.2 and 22.6 min, respectively. The product exhibits the same elution pattern as the ␣1-6FucT of porcine liver (28). The reaction mixture without GDP-Fuc was also subjected to HPLC. Compared with the elution pattern of the reaction mixture with GDP-Fuc, no enzymatic product was detected in the absence of GDP-Fuc in the reaction mixture. To confirm the structure of the product, the reaction was performed on a large scale, and the enzymatic product was analyzed with an ion spray mass spectrophotometer. The substrate, GnGn-bi-Asn-PABA, has a molecular weight of 1576. In contrast, the enzymatic product has one of 1725. This indicated that this ␣1-6FucT catalyzes the transfer of 1 mol of fucose to 1 mol of GnGn-bi-Asn-PABA (data not shown). The chemical shift value of the enzymatic product on 1 H NMR analysis was the same as synthesized GnGnF-bi-Asn-PABA, which had previously been published (28). The enzymatic product gave the two typical signals of fucose at ␦ ϭ 4.864 ppm (H-1) and ␦ ϭ 1.193 ppm (CH3) ( Table III, boldface type).
The purified enzyme was incubated under various conditions. The pH optimum was 7.0 (Fig. 4), and divalent cations such as Mg 2ϩ and Ca 2ϩ were found to have negligible effects on the activity. This enzyme was fully active in the presence of 5 mM EDTA (Fig. 4). In the presence of 5 mM EDTA at pH 7.0, the K m values for GnGn-bi-Asn-PABA and GDP-Fuc were 25.0 and 46 M, respectively.

Large Scale Purification and Column Chromatography of ␣1-6FucT
100 g of porcine brain was homogenized one time, and then extraction and purification of the enzyme were performed according to the method described in steps 1-3. These steps were repeated 20 times for approximately 2 kg of porcine brain, and the eluate from step 3 was collected and pooled in 20 mM potassium phosphate, pH 7.0, containing 1 M KCl, 5 mM EDTA, and 0.1% Triton X-100. In this solution, ␣1-6FucT is fully stable. The pooled fractions were desalted against 20 mM potassium phosphate, pH 7.0, containing 50 mM KCl, and 5 mM EDTA, using a YM30 membrane, and step 3 was performed again for the total pooled fractions. The eluted fractions were collected and desalted against 20 mM potassium phosphate, pH 7.0, containing 50 mM KCl, and 5 mM EDTA, and then applied to a GDP-hexanolamine-Sepharose 4B column (16 ϫ 5 cm;  Pharmacia HR16/10), which had been equilibrated with 20 mM potassium phosphate buffer containing 50 mM KCl, 5 mM EDTA, and 0.05% Triton X-100. After the column had been washed with the same buffer sufficiently, enzymes were eluted with 20 mM potassium phosphate buffer containing 50 mM KCl, 5 mM EDTA, 0.01% Triton X-100, and 5 mM GDP. The eluted fractions were collected and used for further experiments.

SDS-PAGE and Isoelectric Focusing of Purified ␣1-6FucT
The most purified fraction obtained on a GDP-hexanolamine affinity column migrates as one major component, 58 kDa, on SDS-PAGE under nonreducing conditions, and under reducing conditions the molecular mass of this component changes from 58 to 54 kDa (Fig. 5). The same component gave a diffuse band on isoelectric focusing at pI 7.0, and ␣1-6FucT activity was detected in the gel slices corresponding to this band (Fig. 6).

cDNA Cloning of ␣1-6FucT
The N-terminal sequence determined from a component of 54 kDa on SDS-PAGE was used to design sense strand PCR primers. The internal peptide sequence was determined from four lysyl endopeptidase-derived peptides (Fig. 7) and was used to design several antisense strand PCR primers. The five transcript-specific oligonucleotides obtained were used to amplify a cDNA that encodes the ␣1-6FucT (Fig. 7). Agarose gel electrophoresis of the PCR products demonstrated that antisense primer A12 produced a 1.45-kilobase pair DNA fragment (Fig.  8). The PCR product, S-A12, was then subcloned into pT7 Blue-Vector (Novagen), and the full-length sequence was determined (data not shown). From a gt11 cDNA library of porcine brain (Clonetech), cDNA encoding ␣1-6FucT was also isolated, utilizing the PCR product as a probe. Five positive clones, C1, C2, C3, C4, and C5, were obtained as described in Fig. 8. Clones C1 and C2 appeared to contain the full-length ␣1-6FucT open reading frame, and the nucleotide sequence was determined from these two clones. The DNA sequence determined from the PCR product, S-A12, was completely identical to that of cDNA clone isolated from the gt11 cDNA library.
The translated sequence of ␣1-6FucT is shown in Fig. 9A. The 1,728-base pair open reading frame encodes a 575-amino acid polypeptide. The deduced amino acid composition was identical to that of the purified enzyme determined by amino acid analysis. Both the N-terminal and internal peptide amino acid sequences are included within the predicted sequence, as underlined in Fig. 9A.

Transient Expression of ␣1-6FucT in COS-1 Cells
To verify that the cloned cDNA encodes ␣1-6FucT transferase, the coding region of cDNA was subcloned into the mammalian expression vector, pSVK3, and then the vector was transfected into COS-1 cells. After 48 h, the cells were harvested, and ␣1-6FucT activity was measured in the homogenate. Preliminary experiments indicated that untransfected COS-1 cells show very little enzyme activity. COS-1 cells transfected with an expression vector containing ␣1-6FucT showed high activity, i.e. 2,360 pmol/h/mg of protein, compared with the cells transfected with the control plasmid, mock pSVK3 (Table IV).

Northern Blotting Analysis of Total RNA from Rat Brain
Northern blotting analysis of total RNA from rat brain showed a band at 3.5 kilobases (Fig. 10). Brain showed one of the highest activities among various tissues, and the content of ␣1-6FucT mRNA of brain was also high. DISCUSSION We have reported the purification of N-acetylglucosaminyltransferases III and V (31,32) and employed donor and acceptor substrates for the sequential affinity chromatographies. In this paper we found that these chromatographies using donor and acceptor substrates as ligand were fairly effective for purification of ␣1-6FucT from porcine brain. Electrophoresis of the purified ␣1-6FucT gave a diffuse single band on the isoelectric focusing at pI 7.0 (Fig. 6). Based on the deduced amino acid composition from the cDNA sequence, the pI is 7.39. No putative N-glycosylation sites were found like several glycosyltransferases (33). On SDS-PAGE, a major band of 58 kDa appeared under nonreducing conditions. On reduction, however, the molecular mass changed to 54 kDa. Because of the limited material available for our study, the mechanism by which this change occurs is still unknown.
Weinstein et al. (34) reported that the amino terminus of glycosyltransferase was subjected to proteolytic cleavage due to an endogenous protease (32). According to the results of amino acid analysis, the cleavage site is very strict in the case of the N terminus of ␣1-6FucT as in the case of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (35). It seems that the enzyme was cleaved by an endogenous protease during the extraction procedure.
No significant homology was found with previously cloned ␣FucTs (36). But this enzyme has a domain structure similar to those of other glycosyltransferases (33,37); ␣1-6FucT also has a short amino-terminal cytoplasmic tail, a 16-amino acid transmembrane sequence, a "stem" region, and a long C-terminal catalytic part, which is in the Golgi lumen (38). This enzyme has a proline-rich domain, 10 proline residues being found among residues 261-340 (31,39). Based on the hydropathy analysis, a hydrophobic region of 16 amino acids precedes the amino acid terminus and is likely to be the transmembrane region of this enzyme (Fig. 9B).
␣1-6FucT from a human fibroblast cell line required the Mg 2ϩ cation and did not bind to a GDP-hexanolamine column (27). However, ␣1-6FucT of human serum required no cations for its activity and bound tightly to a GDP-hexanolamine column (28). ␣1-6FucT in porcine liver has an optimum pH of 5.6 (23,28). In contrast, ␣1-6FucT in porcine brain has an opti-  mum pH 7.0 (Fig. 4). Lin, A. I. et al. (40) have reported a novel ␣1-6FucT that can catalyze the transfer of fucose to high mannose type oligosaccharide. And these results suggest that at least two kinds of ␣1-6FucTs may exist and that ␣1-6FucT gene families may exist as observed in the ␤1-6GnT, ␣1-3FucT, and sialyltransferase gene families (41)(42)(43).
Although fucose attached at the nonreducing end of GlcNAc has been well analyzed and the interaction between the sialyl Lewis X and selectin families plays a significant role in the migration and homing of hematopoetic cells, little is known about the function of ␣1-6fucose attached to the reducing end of GlcNAc. In plants, the sugar chains of glycoproteins play an important role in cell-cell recognition signals for multicellular organs, and lectins are their partners in this event. Legume lectins serve as mediators for the synthetic interactions between plants and nitrogen-fixing microorganisms, an important process in the nitrogen cycle (44), and ␣1-6fucose linked to N-acetylglucosamine (GlcNAc-1) greatly enhances the reactivity of legume lectins of the Viciae tribe toward N-linked type oligosaccharides (45). It is supposed that ␣1-6-linked fucose, which is catalyzed by ␣1-6FucT, may play an important role in the regulation of intracellular interactions. In animal tissues, it has been reported that fucose-containing glycoproteins, such as lactoferrin, are cleared rapidly from the blood into the liver (46), and recently mannose/L-fucose-mediated clearance was also proved in alligator liver (47). And it is generally known that a fucose/mannose receptor exists on the surface of macrophages, which is different from asialoglycoprotein receptors, and mediates phagocytosis (48). Recently, there is speculation that MRF plays a role in protection from parasites or fungi (49,50), in addition to its scavenging activity toward aging neutrophils in inflammation (51).
␣1-6FucT catalyzes the transfer of fucose to asparaginelinked GlcNAc in an early step of the processing of N-linked complex type glycopeptides. Since N-linked glycosylation proceeds in a stepwise manner, it is supposed that the addition of fucose to asparagine-linked GlcNAc determines the posttranslational processing and the function of a glycopeptide (52)(53)(54). Recently, mannose-binding mammalian lectins, such as VIP36 and ERGIC-53 (MR 60), were found to exhibit homology to legume lectins and were supposed to regulate the secretion of N-glycans (55)(56)(57). ␣1-6FucT was presumed to be localized in the medial Golgi apparatus, and there is the possibility that attachment of ␣-fucose to asparagine-linked GlcNAc regulates the interaction between intrinsic lectins and N-linked glycoproteins. On the other hand, the commercial endo-␤-Nacetylglucosaminidase F peptidase N-glycosidase/glycanyl amidase (PNGase) mixture readily cleaves high mannose and oligosaccharides with a common core ␣1-6-linked fucose found in thyroglobulin; however, the same endo-␤-N-acetylglucosaminidase F mixture does not cleave the nonfucosylated complex oligosaccharide found in human transferrin (44). ␣-Fucose is presumed to play a role in N-glycan cycle and to determine the function of glycoproteins. FIG. 10. Northern blotting analyses of total RNA form rat brain. Total RNA from rat brain showed a faint band at 3.5 kilobases (upper panel). Ethidium bromide staining shows a comparable amount of RNA in each lane (lower panel)