title: Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms.
abstract: In addition to soluble acid hydrolases, many nonlysosomal proteins have been shown to bear mannose 6-phosphate (Man-6-P) residues. Quantification of the extent of mannose phosphorylation and the relevance to physiological function, however, remain poorly defined. In this study, we investigated the mannose phosphorylation status of leukemia inhibitory factor (LIF), a previously identified high affinity ligand for the cation-independent mannose 6-phosphate receptor (CI-MPR), and we analyzed the effects of this modification on its secretion and uptake in cultured cells. When media from LIF-overexpressing cells were fractionated using a CI-MPR affinity column, 35-45% of the total LIF molecules were bound and specifically eluted with free Man-6-P thus confirming LIF as a bona fide Man-6-P-modified protein. Surprisingly, mass spectrometric analysis of LIF glycopeptides enriched on the CI-MPR column revealed that all six N-glycan sites could be Man-6-P-modified. The relative utilization of these sites, however, was not uniform. Analysis of glycan-deleted LIF mutants demonstrated that loss of glycans bearing the majority of Man-6-P residues leads to higher steady-state levels of secreted LIF. Using mouse embryonic stem cells, we showed that the mannose phosphorylation of LIF mediates its internalization thereby reducing extracellular levels and stimulating embryonic stem cell differentiation. Finally, immunofluorescence experiments indicate that LIF is targeted directly to lysosomes following its biosynthesis, providing another mechanism whereby mannose phosphorylation serves to control extracellular levels of LIF. Failure to modify LIF in the context of mucolipidosis II and its subsequent accumulation in the extracellular space may have important implications for disease pathogenesis.
authors: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
authors: Jarrod
authors: J
authors: Barnes
authors: Jae-Min
authors: JM
authors: Lim
authors: Anne
authors: A
authors: Godard
authors: Frédéric
authors: F
authors: Blanchard
authors: Lance
authors: L
authors: Wells
authors: Richard
authors: R
authors: Steet
references: Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8439-44
references: 10900005
references: J Proteome Res. 2008 Jul;7(7):3010-21
references: 18507433
references: Nature. 2009 Jul 2;460(7251):118-22
references: 19571885
references: Cytokine. 2009 Jun;46(3):332-8
references: 19342253
references: J Biol Chem. 1999 Jul 2;274(27):19309-15
references: 10383441
references: Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):17125-9
references: 16286643
references: Mol Cell Proteomics. 2006 Oct;5(10):1942-56
references: 16709564
references: Am J Respir Cell Mol Biol. 1999 Apr;20(4):834-41
references: 10101017
references: Eur J Immunol. 1999 Mar;29(3):1004-13
references: 10092105
references: Trends Mol Med. 2010 Nov;16(11):493-500
references: 20870461
references: Nat Rev Rheumatol. 2011 Jan;7(1):33-42
references: 21119608
references: Curr Immunol Rev. 2010 Aug 1;6(3):167-173
references: 21037937
references: J Cell Biol. 1987 Nov;105(5):1947-55
references: 2960682
references: J Biol Chem. 1997 Aug 1;272(31):19408-12
references: 9235940
references: Endocrinology. 2010 Apr;151(4):1713-22
references: 20181800
references: BMB Rep. 2010 Aug;43(8):513-23
references: 20797312
references: FASEB J. 1987 Dec;1(6):462-8
references: 3315809
references: J Biol Chem. 2002 Aug 16;277(33):29737-44
references: 12058031
references: Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):580-4
references: 1846448
references: J Biol Chem. 2010 Jul 30;285(31):23936-44
references: 20489197
references: Mol Cell Proteomics. 2006 Apr;5(4):686-701
references: 16399764
references: J Biol Chem. 2009 Feb 6;284(6):3777-83
references: 19075021
references: J Biol Chem. 1981 Dec 10;256(23):11977-80
references: 6457829
references: J Biol Chem. 1988 Oct 5;263(28):14211-5
references: 2971654
references: Mol Cells. 2011 Feb;31(2):181-9
references: 21191816
references: Cytokine. 2000 Jun;12(6):613-21
references: 10843736
references: J Clin Invest. 2002 Jan;109(2):269-76
references: 11805139
references: J Clin Invest. 1993 Apr;91(4):1575-81
references: 8473500
references: J Biol Chem. 1988 Mar 5;263(7):3521-7
references: 2963825
references: Cell. 2005 Dec 29;123(7):1307-21
references: 16377570
references: J Biol Chem. 1992 Feb 15;267(5):3214-22
references: 1737777
references: J Clin Invest. 1992 Sep;90(3):888-96
references: 1522240
references: J Clin Invest. 2000 Mar;105(5):673-81
references: 10712439
references: Mol Biol Cell. 2001 Jun;12(6):1623-31
references: 11408573
references: Clin Exp Metastasis. 2009;26(2):133-41
references: 18953658
references: Arch Biochem Biophys. 1992 Mar;293(2):349-55
references: 1536571
references: Cell Struct Funct. 2008;33(1):1-12
references: 18256512
references: J Biol Chem. 1992 Nov 15;267(32):23342-8
references: 1331081
references: J Biol Chem. 1998 Aug 14;273(33):20886-93
references: 9694835
references: Am J Physiol. 1994 Apr;266(4 Pt 1):L426-35
references: 8179019
references: J Biol Chem. 1989 Mar 25;264(9):5015-23
references: 2925680
references: Wound Repair Regen. 2000 Nov-Dec;8(6):538-46
references: 11208181
references: J Biol Chem. 1992 Nov 15;267(32):23349-56
references: 1331082
references: J Biol Chem. 1995 Jan 6;270(1):170-9
references: 7814370
references: Mol Biol Cell. 2006 May;17(5):2312-21
references: 16510524
references: J Biol Chem. 1999 Aug 27;274(35):24685-93
references: 10455136
references: Glycobiology. 2008 Sep;18(9):664-78
references: 18621992
references: Arch Biochem Biophys. 1991 May 1;286(2):562-8
references: 1897978
references: J Biol Chem. 2002 Nov 1;277(44):41897-905
references: 12202476
references: J Org Chem. 2005 Nov 25;70(24):9892-904
references: 16292820
references: Proc Natl Acad Sci U S A. 2010 Jul 13;107(28):12493-8
references: 20615935
references: J Biol Chem. 1998 Aug 14;273(33):21067-76
references: 9694859
references: J Biol Chem. 2005 Sep 30;280(39):33318-23
references: 16081416
references: J Biol Chem. 2003 Aug 15;278(33):31226-32
references: 12782633
references: Biochemistry. 2007 Nov 6;46(44):12604-17
references: 17927214
references: Cytokine. 2002 Dec 7;20(5):224-30
references: 12550107
references: J Med Genet. 2010 Jan;47(1):38-48
references: 19617216
references: Stem Cells. 2003;21(1):5-14
references: 12529546
references: J Proteome Res. 2009 Feb;8(2):673-80
references: 19072240
references: J Biol Chem. 2010 Feb 19;285(8):5907-16
references: 20022960