N-Glycosylation of Laminin-332 Regulates Its Biological Functions
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m804526200
ISSN1083-351X
AutoresYoshinobu Kariya, Rika Kato, Satsuki Itoh, Tomohiko Fukuda, Yukinao Shibukawa, Noriko Sanzen, Kiyotoshi Sekiguchi, Yoshinao Wada, Nana Kawasaki, Jianguo Gu,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoLaminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of β1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and β1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired α3β1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer. Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of β1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and β1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired α3β1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer. Laminins (Lms) 2The abbreviations used are: Lm, laminin; BRL, buffalo rat liver; BSA, bovine serum albumin; CBB, Coomassie Brilliant Blue; ECM, extracellular matrix; E4-PHA, erythroagglutinating phytohemagglutinin; ELISA, enzyme-linked immunosorbent assay; GnT-III, N-acetylglucosaminyltransferase III; GnT-V, N-acetylglucosaminyltransferase V; LC, liquid chromatography; L4-PHA, leukoagglutinating phytohemagglutinin; Lmxyz, laminin-xyz; MS, mass spectrometry; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. are large heterotrimeric glycoproteins that are prominent components of basement membranes and are involved in important biological roles, including tissue development and cell differentiation, survival, adhesion, and migration (1Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1058) Google Scholar). Lms are heavily glycosylated molecules. It has been reported that between 13 and 30% of the total molecular weight of Lms is N-linked glycosylated (2Fujiwara S. Shinkai H. Deutzmann R. Paulsson M. Timpl R. Biochem. J. 1988; 252: 453-461Crossref PubMed Scopus (104) Google Scholar). Laminin-111 (Lm111; previously known as laminin-1) is easily purified from mouse Engelbreth-Horm-Swarm tumor and has been intensively investigated for its carbohydrate structures. In comparison with unglycosylated Lm111, which is purified from cell lysates of tunicamycin-treated cells, glycosylation of Lm111 was shown to affect cell spreading and neurite outgrowth activities but not cell adhesion activity or heterotrimer assembly (3Dean III, J.W. Chandrasekaran S. Tanzer M.L. J. Biol. Chem. 1990; 265: 12553-12562Abstract Full Text PDF PubMed Google Scholar, 4Green T.L. Hunter D.D. Chan W. Merlie J.P. Sanes J.R. J. Biol. Chem. 1992; 267: 2014-2022Abstract Full Text PDF PubMed Google Scholar). However, tunicamycin extensively inhibited the secretion of laminin into cell culture medium (3Dean III, J.W. Chandrasekaran S. Tanzer M.L. J. Biol. Chem. 1990; 265: 12553-12562Abstract Full Text PDF PubMed Google Scholar, 5Howe C.C. Mol. Cell Biol. 1984; 4: 1-7Crossref PubMed Scopus (28) Google Scholar). Laminin-332 (Lm332; previously known as laminin-5) is composed of α3, β3, and γ2 chains. Lm332 is expressed in the skin and other stratified squamous epithelial tissues, where is associate with hemidesmosomes via integrin α6β4. Genetic absence of Lm332 causes a severe and lethal skin blistering disease, Herlitz's junctional epidermolysis bullosa (6Carter W.G. Ryan M.C. Gahr P.J. Cell. 1991; 65: 599-610Abstract Full Text PDF PubMed Scopus (685) Google Scholar, 7Ryan M.C. Christiano A.M. Engvall E. Wewer U.M. Miner J.H. Sanes J.R. Burgeson R.E. Matrix Biol. 1996; 15: 369-381Crossref PubMed Scopus (119) Google Scholar). In vitro Lm332 promotes cell motility and scattering through the association of C-terminal globular (G) domains with integrin α3β1 (8Kariya Y. Tsubota Y. Hirosaki T. Mizushima H. Puzon-McLaughlin W. Takada Y. Miyazaki K. J. Cell Biochem. 2003; 88: 506-520Crossref PubMed Scopus (39) Google Scholar), which is thought to be a key factor during wound healing (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 10Kariya Y. Miyazaki K. Exp. Cell Res. 2004; 297: 508-520Crossref PubMed Scopus (57) Google Scholar) and cancer metastasis (11Miyazaki K. Cancer Sci. 2006; 97: 91-98Crossref PubMed Scopus (148) Google Scholar, 12Marinkovich M.P. Nat. Rev. Cancer. 2007; 7: 370-380Crossref PubMed Scopus (244) Google Scholar). Proteolytic cleavage of the Lm332 α3 and γ2 subunits (13Giannelli G. Falk-Marzillier J. Schiraldi O. Stetler-Stevenson W.G. Quaranta V. Science. 1997; 277: 225-228Crossref PubMed Scopus (1051) Google Scholar, 14Veitch D.P. Nokelainen P. McGowan K.A. Nguyen T.T. Nguyen N.E. Stephenson R. Pappano W.N. Keene D.R. Spong S.M. Greenspan D.S. Findell P.R. Marinkovich M.P. J. Biol. Chem. 2003; 278: 15661-15668Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) affects both cell adhesion and migration (15Koshikawa N. Giannelli G. Cirulli V. Miyazaki K. Quaranta V. J. Cell Biol. 2000; 148: 615-624Crossref PubMed Scopus (562) Google Scholar, 16Tsubota Y. Yasuda C. Kariya Y. Ogawa T. Hirosaki T. Mizushima H. Miyazaki K. J. Biol. Chem. 2005; 280: 14370-14377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Few studies have assessed the functional significance of N-glycosylation of Lm332 subunits. N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of the β1,6-linked GlcNAc branch and defines this subset of N-glycans (17Cummings R.D. Trowbridge I.S. Kornfeld S. J. Biol. Chem. 1982; 257: 13421-13427Abstract Full Text PDF PubMed Google Scholar, 18Shoreibah M. Perng G.S. Adler B. Weinstein J. Basu R. Cupples R. Wen D. Browne J.K. Buckhaults P. Fregien N. J. Biol. Chem. 1993; 268: 15381-15385Abstract Full Text PDF PubMed Google Scholar) (Fig. 1A). In some cancers, an increase of β1,6-GlcNAc is related to cancer metastasis. This is supported by several reports, including GnT-V overexpression in cancer (19Dennis J.W. Laferte S. Cancer Res. 1989; 49: 945-950PubMed Google Scholar, 20Handerson T. Pawelek J.M. Cancer Res. 2003; 63: 5363-5369PubMed Google Scholar) and GnT-V-deficient mouse studies (21Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (481) Google Scholar). By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) (Fig. 1A) suppresses further processing with branching enzymes, such as GnT-V, and elongation of N-glycans (22Gu J. Nishikawa A. Tsuruoka N. Ohno M. Yamaguchi N. Kangawa K. Taniguchi N. J. Biochem. (Tokyo). 1993; 113: 614-619Crossref PubMed Scopus (139) Google Scholar, 23Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (497) Google Scholar), resulting in down-regulating cancer metastasis (24Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8754-8758Crossref PubMed Scopus (260) Google Scholar). In addition, GnT-III modification of α3β1 integrin inhibits cell migration promoted by GnT-V on the Lm332 substrate (25Zhao Y. Nakagawa T. Itoh S. Inamori K. Isaji T. Kariya Y. Kondo A. Miyoshi E. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 32122-32130Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In the present study, to investigate the effect of N-glycosylation on Lm332 function, we focused on GnT-III- and GnT-V-mediated N-glycosylation of Lm332. Therefore, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. The analysis of lectin blotting and mass spectrometry indicated that Lm332 could be modified by either GnT-III or GnT-V. As a result, GnT-III modification of Lm332 caused a decrease in its keratinocyte cell adhesion and migration activities. Our findings demonstrate a novel regulatory mechanism of Lm332 activities brought on by N-glycosylation. Antibodies and Reagents—Mouse monoclonal antibodies against the N-terminal regions of the human laminin α3 chain (Lsαc3) and the γ2 chain (D4B5) were a generous gift from Dr. Kaoru Miyazaki (Yokohama City University, Yokohama, Japan). Mouse monoclonal antibody against the laminin α3 chain (2B10) was prepared as previously described (26Fujiwara H. Kikkawa Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 17550-17558Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Monoclonal antibodies against the human laminin β3 chain (kalinin B1) and paxillin were purchased from Transduction Laboratories (Lexington, KY). Control mouse and rat IgG and function-blocking anti-integrin α3 (P1B5) and α6 (GoH3) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Alexa Fluor 488 goat anti-mouse IgG was obtained from Invitrogen. Biotinylated leukoagglutinating phytohemagglutinin (L4-PHA) and biotinylated erythroagglutinating phytohemagglutinin (E4-PHA) were from Seikagaku Biobusiness Corp. (Tokyo, Japan). A monoclonal antibody against α-tubulin was purchased from Sigma. Cell Culture—The human gastric cancer cell line MKN45 was cultured in RPMI 1640 medium (Nacalai Tesque, Japan). MKN45 transfectants were described previously (27Ihara S. Miyoshi E. Ko J.H. Murata K. Nakahara S. Honke K. Dickson R.B. Lin C.Y. Taniguchi N. J. Biol. Chem. 2002; 277: 16960-16967Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The Buffalo rat liver-derived epithelial cell line BRL was a gift from Dr. Kaoru Miyazaki (Yokohama City University, Yokohama, Japan) and was maintained in Dulbecco's modified Eagle's medium. Those media were supplemented with 10% fetal calf serum, penicillin, and streptomycin sulfate. Keratinocytes isolated from patients with junctional epidermolysis bullosa lacking Lm332 were a generous gift from Dr. M. Peter Marinkovich (Stanford University). Keratinocytes were grown in 50% defined keratinocyte medium (Invitrogen) and 50% medium 154 (Cascade Biologics, Portland, OR) containing penicillin and streptomycin sulfate. Preparation of Conditioned Medium and Purification of Laminin-332—For purification of Lm332, the serum-free CM from MKN45 transfectants were collected every 2 or 3 days. The collected media were centrifuged at 1,000 rpm for 10 min. Finally its supernatant was collected and used as a source for purification of Lm332. The protein containing the supernatant was precipitated by 80% saturated ammonium sulfate. The precipitate was dissolved in and dialyzed against a gelatin column buffer (20 mm Tris-HCl (pH 7.5), 0.1 m NaCl, 0.1% CHAPS, 0.005% Brij 35) overnight at 4 °C. Then samples were centrifuged at 19,000 rpm for 30 min at 4 °C to remove the undissolved proteins. The precleared solution was passed through a gelatin column, and then its flow-through was directly applied to an α3 antibody (2B10) column. After washing with the antibody column buffer (20 mm Tris-HCl (pH 7.5), 0.5 m NaCl, 0.1% CHAPS, 0.005% Brij 35), followed by MilliQ water, binding proteins were eluted by 0.05% trifluoroacetic acid (v/v) and immediately neutralized with Tris-HCl (pH 8.0) containing 0.005% Brij 35 and 0.1% CHAPS. Preparation of Cell Lysate—For preparing cell lysate, cells were washed with cold PBS twice and then lysed with lysis buffer (1% Triton X-100, 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA) containing protease inhibitor mixture (Nacalai Tesque). After incubation for 10 min on ice, cell lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4 °C, and its supernatants were used in the following samples. The protein concentration was determined using a protein assay kit (Nacalai Tesque). SDS-PAGE, Immunoblotting, and Lectin Blotting—SDS-PAGE was performed either on 6% gels or on 4.0–7.5% gradient gels under reducing or nonreducing conditions. Separated proteins were stained with Coomassie Brilliant Blue (CBB). ImageJ software was used for densitometric analysis of protein bands. For immunoblotting analyses, proteins resolved by SDS-PAGE were transferred to nitrocellulose membranes. The blots were probed with each specific antibody or biotinylated E4-PHA and L4-PHA. Immunoreactive bands were detected using an ECL detection kit (GE Healthcare) and a Vectastain ABC kit (Vector Laboratories). Analysis of N-Glycan Structures by Liquid Chromatography/Multiple-stage Mass Spectrometry (LC/MSn)—Purified Lm332 was applied to SDS-PAGE using a 4–7.5% gradient gel under reducing conditions and visualized by CBB staining. The gel bands corresponding to α3, β3, and γ2 subunits were excised from the gel and then cut into pieces, respectively. The gel pieces were destained with 25 mm NH4HCO3 containing 30% acetonitrile and then dehydrated with 100% acetonitrile. The proteins in the gel were reduced and carboxymethylated by incubation with dithiothreitol and sodium monoiodoacetate (25Zhao Y. Nakagawa T. Itoh S. Inamori K. Isaji T. Kariya Y. Kondo A. Miyoshi E. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 32122-32130Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). N-Glycans were released by the treatment with peptide:N-glycanase F, and extracted from the gel pieces, as reported (28Kuster B. Wheeler S.F. Hunter A.P. Dwek R.A. Harvey D.J. Anal. Biochem. 1997; 250: 82-101Crossref PubMed Scopus (325) Google Scholar). The oligosaccharides were reduced with sodium borohydride and desalted. LC/MSn was performed using a quadropole linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (Finnigan LTQ-FT™; Thermo Fisher Scientific Corp., San Jose, CA) connected to a nanoflow LC system (NanoFrontier nLC; Hitachi High-Technologies Corp., Japan). The eluents were 5 mm ammonium acetate containing 2% acetonitrile, pH 9.6 (pump A), and 5 mm ammonium acetate containing 80% acetonitrile, pH 9.6 (pump B). The borohydride-reduced N-glycans were separated on a Hypercarb column (0.075 × 150 mm; Thermo Fisher Scientific Corp.) with a linear gradient of 5–35% of pump B in 110 min. A single mass scan with Fourier transform (m/z 450–2,000) followed by data-dependent MS/MS for the most intense ions was performed in both positive and negative ion modes as previously reported (29Itoh S. Kawasaki N. Hashii N. Harazono A. Matsuishi Y. Hayakawa T. Kawanishi T. J. Chromatogr. A. 2006; 1103: 296-306Crossref PubMed Scopus (33) Google Scholar). Cell Adhesion Assay—Cell adhesion assay was performed as described previously (8Kariya Y. Tsubota Y. Hirosaki T. Mizushima H. Puzon-McLaughlin W. Takada Y. Miyazaki K. J. Cell Biochem. 2003; 88: 506-520Crossref PubMed Scopus (39) Google Scholar). Briefly, each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate (Costar, Cambridge, MA) was coated with a substrate protein and then blocked with 1% bovine serum albumin (BSA). 2 × 104 cells in supplement-free keratinocyte growth medium were inoculated per well of 96-well plates. After nonadherent cells were removed, adherent cells were fixed with 25% (w/v) glutaraldehyde and stained with 0.5% crystal violet (w/v) in 20% (v/v) methanol for 10 min. The well was measured for absorbance at 590 nm using a microplate reader. For inhibition assay, the cell suspension was incubated with function-blocking anti-integrin antibodies or with the control IgG for 20 min at room temperature before inoculation. Cell Spreading Assay—For measurement of the cell-spreading area, keratinocytes on purified Lm332 substrates were incubated in keratinocyte growth medium. After 1 h, at least 100 cells were photographed, and their cell spreading areas were measured using Axio Vision software (Carl Zeiss, Germany). Cell Scattering Assay—A scattering assay was done as reported previously (10Kariya Y. Miyazaki K. Exp. Cell Res. 2004; 297: 508-520Crossref PubMed Scopus (57) Google Scholar). Briefly, 500 μl of cell suspension (2 × 104 cells in Dulbecco's modified Eagle's medium plus 1% (v/v) fetal calf serum) were inoculated per well of 24-well plates. Test samples were directly added into the culture medium and incubated at 37 °C. After 40 h, cells were fixed with 25% (w/v) glutaraldehyde and stained with 0.5% crystal violet (w/v) in 20% (v/v) methanol for 10 min. The scattered single cells were counted, and the degree of cell scattering was expressed as the percentage of single cells in each field. At least 300 cells were counted in each field. Cell Migration Assay—A glass bottom dish (Asahi Techno Glass, Japan) was precoated with purified Lm332 and then blocked with 1% BSA for 1 h at 37°C. 200 μl of Lm332-null keratinocyte cell suspension (4 × 104 cells/ml) in growth medium were inoculated into each Lm332-precoated glass bottom dish. After incubation for 1 h at 37°C, cell movement was monitored using time lapse video equipment (Carl Zeiss) for 8 h. ELISA—The ELISA was as follows. The wells of a 96-well plate were coated with test proteins and then blocked with 1.2% BSA at room temperature for 1 h. The wells were washed with PBS containing 0.05% Tween 20 (washing buffer) three times and then incubated with primary antibody for 1 h at room temperature. Furthermore, the wells were washed with washing buffer three times and then incubated with secondary antibody coupled with biotin for 45 min at room temperature. Similarly, the wells were washed three times and incubated with alkaline phosphatase conjugated with avidin D for 45 min at room temperature. After five washes with washing buffer, the bound antibodies were quantified by their absorbance at 405 nm after incubation with p-nitrophenylphosphate disodium salt in 100 mm diethanolamine (pH 9.8) containing 0.24 mm MgCl2. Immunofluorescence Microscopy—A glass bottom dish (Asahi Techno Glass) was precoated with purified Lm332 and then blocked with 1% BSA for 1 h at 37°C. 200 μl of the cell suspension (2 × 105 cells/ml) in growth medium were inoculated into each Lm332-precoated glass bottom dish. After incubation for 1 h, the cells were washed with PBS and then fixed with 4% (w/v) paraformaldehyde in PBS for 10 min. For permeabilization, the cells were treated with 0.2% (v/v) Triton X-100 in PBS. The fixed cells were blocked with 2% BSA in PBS for 1 h before staining with appropriate primary and secondary antibodies. Fluorescence images were obtained using a fluorescence microscope (Olympus, Tokyo) equipped with ×100/1.35 UPlan-Apochromat oil immersion objectives. Statistical Analysis—Data are presented as mean ± S.D. Student's t test, with Microsoft Excel, was used to compare the two groups. Characterization of GnT-III and GnT-V Transfectants—As a source for purification of Lm332 modified by GnT-III- or GnT-V-catalyzed glycosylation, we prepared GnT-III- and GnT-V-overexpressing MKN45 transfectants and also a vector-only transfectant to be used as a negative control as previously described (27Ihara S. Miyoshi E. Ko J.H. Murata K. Nakahara S. Honke K. Dickson R.B. Lin C.Y. Taniguchi N. J. Biol. Chem. 2002; 277: 16960-16967Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). To check the changes in oligosaccharide structures for each transfectant, lectin blotting was performed against the cell lysates from those transfectants using E4-PHA, which preferentially binds to bisecting GlcNAc residues in N-glycans or L4-PHA, which preferentially binds to β1,6-branched GlcNAc residues (30Cummings R.D. Kornfeld S. J. Biol. Chem. 1982; 257: 11230-11234Abstract Full Text PDF PubMed Google Scholar). The blotting results showed that the GnT-III transfectant increased bisecting GlcNAc compared with the other two (Fig. 1B, E4-PHA) but decreased GnT-V products (Fig. 1B, L4-PHA), supporting the notion that GnT-III antagonizes the action of GnT-V for the modification of some target glycoproteins, such as α3β1 integrin (25Zhao Y. Nakagawa T. Itoh S. Inamori K. Isaji T. Kariya Y. Kondo A. Miyoshi E. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 32122-32130Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). On the other hand, GnT-V overexpression had no effect on the GnT-III product level (Fig. 1B, E4-PHA) but led to a strong increase of β1,6-branched GlcNAc in MKN45 cells (Fig. 1B, L4-PHA). The immunoblotting using an anti-α-tubulin antibody showed that the amount of a loaded protein was almost the same among the three samples (Fig. 1B, α-tubulin). Purification and Characterization of Lm332 from MKN45 Transfectants—To examine whether Lm332 could be modified by GnT-III or GnT-V to regulate its functions, we tried to purify Lm332 from the CM of vector-, GnT-III-, and GnT-V-MKN45 transfectants using a laminin α3 chain antibody (2B10) column. Purified Lm332 was run on 4–7.5% gradient gel and 6% gel for CBB staining and immunoblotting, respectively (Fig. 2). CBB staining under nonreducing conditions revealed two major bands at ∼460 and 440 kDa, which correspond to the Lm332 forms with an unprocessed 150-kDa γ2 chain and with a processed 105-kDa γ2 chain, respectively (Fig. 2, CBB, left). SDS-PAGE under reducing conditions separated the Lm subunits. All purified Lm332s contained the three major bands of 160-, 150-, and 135-kDa proteins, which correspond to the processed α3 chain, the unprocessed γ2 chain, and the β3 chain, respectively (Fig. 2, CBB, right). In addition to those bands, there are three extra bands, which were larger than the 160-kDa processed α3 chain band. Mass spectrometry analysis showed that those bands are laminin β1, γ1, and an unprocessed α3 chain (190 kDa) (data not shown). A likely explanation is that this represented laminin-311 (Lm311; laminin-6), which is composed of α3, β1, and γ1 chains. Lm332 purified from GnT-III MKN45 transfectant (GnT-III-Lm332) contained slightly increased amounts of those extra bands rather than the Lm332 from vector MKN45 (vector-Lm332) and from GnT-V MKN45 transfectants (GnT-V-Lm332), suggesting that the processing of the α3 chain, but not the β3 and γ2 chains, could be affected by the addition of bisecting GlcNAc. The quantified band intensity upon CBB staining under reducing conditions showed that the ratio of β3 chain in Lm332 to β1 chain in Lm311 was 10:1 (data not shown). Under nonreducing conditions, it was difficult to find the bands corresponding to Lm332 with an unprocessed laminin α3 chain (490 kDa) and Lm311 (600 kDa). The compositions of the Lm332 chain were also confirmed by immunoblotting using each chain-specific antibody. Consistent with the results shown in CBB staining, GnT-III-Lm332 contained slightly more unprocessed α3 chains (190-kDa) than those of vector-Lm332 and GnT-V-Lm332 (Fig. 2, I.B. α3). By contrast, there were no differences between the 135-kDa β3 chain, the unprocessed 150-kDa γ2 chain, and the processed 105-kDa γ2 chain among three Lm332s (Fig. 2, I.B. β3 and γ2). To establish whether Lm332 was modified by GnT-III or GnT-V, lectin blotting using E4-PHA and L4-PHA lectin was performed against three purified Lm332s. A comparison of bands corresponding to α3 and β3 chains among three purified Lm332s indicated that increased GnT-III and GnT-V products presented on GnT-III-Lm332 and GnT-V-Lm332, respectively (Fig. 3). Upon E4-PHA lectin blotting against GnT-III-Lm332, bisecting GlcNAc was added to β1 and γ1 chains as well as to the unprocessed α3A chain (Fig. 3, E4-PHA). To ascertain the N-glycan structures on Lm332 subunits directly, we also performed liquid chromatography/multiple-stage mass spectrometry (LC/MSn) analyses. Each subunit of Lm332 was separated by SDS-PAGE under reducing conditions, and N-glycans in each subunit were released by in-gel digestion with peptide:N-glycanase F. The oligosaccharides were extracted from the gel and subjected to LC/MSn as described under "Experimental Procedures." Total ion chromatograms were acquired by single mass scans (m/z 450–2,000) in positive (Fig. 4A) and negative (Fig. 4B) ion modes, respectively. Structures of N-glycans in major peaks were deduced from m/z values of protonated molecules acquired by Fourier transform ion cyclotron resonance-MS and fragment ions in MSn spectra. Additional bisected N-glycans were confirmed based on the fragment ions at m/z 792 ([HexNAc-Hex-Hex-NAc-HexNAc-OH + H]+) and m/z 938 ([HexNAc-Hex-HexNAc-(dHex-)HexNAc-OH + H]+) in MS/MS and MS/MS/MS spectra. The extracted ion chromatograms of representative bisected N-glycans were acquired by single mass scans, and the structures in each peak were deduced (Fig. 4C). The peaks of the bisected N-glycans from all three subunits of GnT-III-Lm332 (Fig. 4, C, b, e, and h) were more intense than those from both the vec-Lm332 (Fig. 4C, a, d, and g) and GnT-V-Lm332 (Fig. 4C, c, f, and i). We also examined the β1,6-GlcNAc structures on all subunits of three Lm332s (Fig. 4D). The majority of N-glycans in all three subunits of vec-Lm332 (Fig. 4D, a, d, and g) and GnT-V-Lm332 (Fig. 4D, c, f, and i) were sialylated triantennary, whereas few triantennary forms were found in those of GnT-III-Lm332 (Fig. 4D, b, e, and h). These results, taken together, suggest that all subunits of Lm332 were modified by GnT-III and GnT-V, and they also support the notion that introduction of GnT-III inhibits GnT-V products.FIGURE 3Lectin blotting analysis of purified laminin-332s. 100 ng of laminin-332s (vector-, GnT-III-, and GnT-V-Lm332) was separated on 6% SDS-PAGE gel and then blotted onto the nitrocellulose membranes. Blotted proteins were probed with E4-PHA (top) and L4-PHA (bottom) lectin. Ordinates indicate molecular sizes in kDa of marker proteins and laminin chains.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Analysis of N-glycan structures of purified laminin-332s by LC/MSn. Total ion chromatograms (TICs) obtained by single mass scans of N-glycans extracted from the gel separated Lm332 subunits (left, α3; middle, β3; right, γ2) of vec-Lm332 (top, vec), GnT-III-Lm332 (middle, GnT-III), and GnT-V-Lm332 (bottom, GnT-V) in positive (A) and negative (B) ion modes. C, extracted ion chromatograms of representative bisected N-glycans acquired by single mass scans. The extracted ion chromatogram of α3 subunit is shown at m/z 822.3, 915.9, 996.9, 1048.9, 1,142.4, and 1,215.5 (a, vector; b, GnT-III-Lm332; c, GnT-V-Lm332). The extracted ion chromatogram of β3 subunit is shown at m/z 1,061.4, 1,142.4, and 1,215.5 (d, vector; e, GnT-III-Lm332; f, GnT-V-Lm332). The extracted ion chromatogram of γ2 subunit is shown at m/z 1,142.4 and 996.9 (g, vector; h, GnT-III-Lm332; i, GnT-V-Lm332). The insets show deduced structures (gray triangle, fucose; open circle, galactose; gray circle, mannose; black square, N-acetylglucosamine; black diamond, N-acetylneuraminic acid). D, integrated mass spectra (m/z 1,000–1,700) acquired at elution positions indicated by boldface lines in total ion chromatograms in negative ion mode (Fig. 4B) (left, α3; middle, β3; right, γ2; top, vector; middle, GnT-III-Lm332; bottom, G
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