Abstract
In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type alpha and type beta have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn-->Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-alpha and FR-beta are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-alpha and FR-beta respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-beta the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-alpha and FR-beta.
Full Text
The Full Text of this article is available as a PDF (406.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Antony A. C. The biological chemistry of folate receptors. Blood. 1992 Jun 1;79(11):2807–2820. [PubMed] [Google Scholar]
- Appel D., Pilarsky C., Graichen R., Koch-Brandt C. Sorting of gp80 (GPIII, clusterin), a marker protein for constitutive apical secretion in Madin-Darby canine kidney (MDCK) cells, into the regulated pathway in the pheochromocytoma cell line PC12. Eur J Cell Biol. 1996 Jun;70(2):142–149. [PubMed] [Google Scholar]
- Bolhuis R. L., Lamers C. H., Goey S. H., Eggermont A. M., Trimbos J. B., Stoter G., Lanzavecchia A., di Re E., Miotti S., Raspagliesi F. Adoptive immunotherapy of ovarian carcinoma with bs-MAb-targeted lymphocytes: a multicenter study. Int J Cancer Suppl. 1992;7:78–81. [PubMed] [Google Scholar]
- Brigle K. E., Spinella M. J., Westin E. H., Goldman I. D. Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells. Biochem Pharmacol. 1994 Jan 20;47(2):337–345. doi: 10.1016/0006-2952(94)90025-6. [DOI] [PubMed] [Google Scholar]
- Brigle K. E., Westin E. H., Houghton M. T., Goldman I. D. Characterization of two cDNAs encoding folate-binding proteins from L1210 murine leukemia cells. Increased expression associated with a genomic rearrangement. J Biol Chem. 1991 Sep 15;266(26):17243–17249. [PubMed] [Google Scholar]
- Canevari S., Mezzanzanica D., Ménard S., Ferrini S., Moretta L., Colnaghi M. I. Possible targets on carcinoma for bMAb retargeting of lymphocyte or drug cytotoxicity. Int J Cancer Suppl. 1992;7:42–44. [PubMed] [Google Scholar]
- Edge C. J., Joao H. C., Woods R. J., Wormald M. R. The conformational effects of N-linked glycosylation. Biochem Soc Trans. 1993 May;21(2):452–455. [PubMed] [Google Scholar]
- Elwood P. C. Molecular cloning and characterization of the human folate-binding protein cDNA from placenta and malignant tissue culture (KB) cells. J Biol Chem. 1989 Sep 5;264(25):14893–14901. [PubMed] [Google Scholar]
- Ferrini S., Cambiaggi A., Sforzini S., Canevari S., Mezzanzanica D., Colnaghi M. I., Moretta L. Use of anti-CD3 and anti-CD16 bispecific monoclonal antibodies for the targeting of T and NK cells against tumor cells. Cancer Detect Prev. 1993;17(2):295–300. [PubMed] [Google Scholar]
- Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
- Ghose-Dastidar J., Ross J. B., Green R. Expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6408–6412. doi: 10.1073/pnas.88.15.6408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Habeck L. L., Leitner T. A., Shackelford K. A., Gossett L. S., Schultz R. M., Andis S. L., Shih C., Grindey G. B., Mendelsohn L. G. A novel class of monoglutamated antifolates exhibits tight-binding inhibition of human glycinamide ribonucleotide formyltransferase and potent activity against solid tumors. Cancer Res. 1994 Feb 15;54(4):1021–1026. [PubMed] [Google Scholar]
- Lacey S. W., Sanders J. M., Rothberg K. G., Anderson R. G., Kamen B. A. Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol. J Clin Invest. 1989 Aug;84(2):715–720. doi: 10.1172/JCI114220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leamon C. P., Low P. S. Cytotoxicity of momordin-folate conjugates in cultured human cells. J Biol Chem. 1992 Dec 15;267(35):24966–24971. [PubMed] [Google Scholar]
- Leamon C. P., Low P. S. Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5572–5576. doi: 10.1073/pnas.88.13.5572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leamon C. P., Low P. S. Selective targeting of malignant cells with cytotoxin-folate conjugates. J Drug Target. 1994;2(2):101–112. doi: 10.3109/10611869409015898. [DOI] [PubMed] [Google Scholar]
- Leamon C. P., Pastan I., Low P. S. Cytotoxicity of folate-Pseudomonas exotoxin conjugates toward tumor cells. Contribution of translocation domain. J Biol Chem. 1993 Nov 25;268(33):24847–24854. [PubMed] [Google Scholar]
- Lee R. J., Low P. S. Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis. J Biol Chem. 1994 Feb 4;269(5):3198–3204. [PubMed] [Google Scholar]
- Luhrs C. A. The role of glycosylation in the biosynthesis and acquisition of ligand-binding activity of the folate-binding protein in cultured KB cells. Blood. 1991 Mar 15;77(6):1171–1180. [PubMed] [Google Scholar]
- Olson T. S., Lane M. D. A common mechanism for posttranslational activation of plasma membrane receptors? FASEB J. 1989 Mar;3(5):1618–1624. doi: 10.1096/fasebj.3.5.2537774. [DOI] [PubMed] [Google Scholar]
- Ratnam M., Marquardt H., Duhring J. L., Freisheim J. H. Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA. Biochemistry. 1989 Oct 3;28(20):8249–8254. doi: 10.1021/bi00446a042. [DOI] [PubMed] [Google Scholar]
- Ross J. F., Chaudhuri P. K., Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer. 1994 May 1;73(9):2432–2443. doi: 10.1002/1097-0142(19940501)73:9<2432::aid-cncr2820730929>3.0.co;2-s. [DOI] [PubMed] [Google Scholar]
- Rothberg K. G., Ying Y. S., Kolhouse J. F., Kamen B. A., Anderson R. G. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol. 1990 Mar;110(3):637–649. doi: 10.1083/jcb.110.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scheiffele P., Peränen J., Simons K. N-glycans as apical sorting signals in epithelial cells. Nature. 1995 Nov 2;378(6552):96–98. doi: 10.1038/378096a0. [DOI] [PubMed] [Google Scholar]
- Shen F., Ross J. F., Wang X., Ratnam M. Identification of a novel folate receptor, a truncated receptor, and receptor type beta in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue specificity. Biochemistry. 1994 Feb 8;33(5):1209–1215. doi: 10.1021/bi00171a021. [DOI] [PubMed] [Google Scholar]
- Shen F., Wu M., Ross J. F., Miller D., Ratnam M. Folate receptor type gamma is primarily a secretory protein due to lack of an efficient signal for glycosylphosphatidylinositol modification: protein characterization and cell type specificity. Biochemistry. 1995 Apr 25;34(16):5660–5665. doi: 10.1021/bi00016a042. [DOI] [PubMed] [Google Scholar]
- Slieker L. J., Martensen T. M., Lane M. D. Synthesis of epidermal growth factor receptor in human A431 cells. Glycosylation-dependent acquisition of ligand binding activity occurs post-translationally in the endoplasmic reticulum. J Biol Chem. 1986 Nov 15;261(32):15233–15241. [PubMed] [Google Scholar]
- Turek J. J., Leamon C. P., Low P. S. Endocytosis of folate-protein conjugates: ultrastructural localization in KB cells. J Cell Sci. 1993 Sep;106(Pt 1):423–430. doi: 10.1242/jcs.106.1.423. [DOI] [PubMed] [Google Scholar]
- Urban J., Parczyk K., Leutz A., Kayne M., Kondor-Koch C. Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line. J Cell Biol. 1987 Dec;105(6 Pt 1):2735–2743. doi: 10.1083/jcb.105.6.2735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993 Apr;3(2):97–130. doi: 10.1093/glycob/3.2.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang X., Shen F., Freisheim J. H., Gentry L. E., Ratnam M. Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates. Biochem Pharmacol. 1992 Nov 3;44(9):1898–1901. doi: 10.1016/0006-2952(92)90089-2. [DOI] [PubMed] [Google Scholar]
- Yan W., Ratnam M. Preferred sites of glycosylphosphatidylinositol modification in folate receptors and constraints in the primary structure of the hydrophobic portion of the signal. Biochemistry. 1995 Nov 7;34(44):14594–14600. doi: 10.1021/bi00044a039. [DOI] [PubMed] [Google Scholar]