Nutrient-responsive O-GlcNAcylation dynamically modulates the secretion of glycan-binding protein galectin 3

Abstract

Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans and that are secreted by a poorly characterized nonclassical secretory mechanism. Once outside the cell, galectins bind to the terminal galactose residues of cell surface glycans and modulate numerous extracellular functions, such as clathrin-independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal; and as we have shown, galectin 3 is a substrate for O-GlcNAc transferase. In this study, we also show that galectin 3 secretion is sensitive to changes in O-GlcNAc levels. We determined using immunoprecipitation and Western blotting that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions, which were dependent on dynamic O-GlcNAcylation. Importantly, we showed that these O-GlcNAc-driven alterations in galectin 3 secretion also facilitated changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function in transducing nutrient-sensing information coded in cell surface glycosylation into biological effects. Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans and that are secreted by a poorly characterized nonclassical secretory mechanism. Once outside the cell, galectins bind to the terminal galactose residues of cell surface glycans and modulate numerous extracellular functions, such as clathrin-independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal; and as we have shown, galectin 3 is a substrate for O-GlcNAc transferase. In this study, we also show that galectin 3 secretion is sensitive to changes in O-GlcNAc levels. We determined using immunoprecipitation and Western blotting that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions, which were dependent on dynamic O-GlcNAcylation. Importantly, we showed that these O-GlcNAc-driven alterations in galectin 3 secretion also facilitated changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function in transducing nutrient-sensing information coded in cell surface glycosylation into biological effects. Nutrient sensing is an essential function of glycosylation (1Wells L. Vosseller K. Hart G. A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance.Cell. Mol. Life Sci. 2003; 60: 222-228Google Scholar, 2Wellen K.E. Thompson C.B. Cellular metabolic stress: Considering how cells respond to nutrient excess.Mol. Cell. 2010; 40: 323-332Google Scholar, 3Zachara N.E. Hart G.W. O-GlcNAc a sensor of cellular state: The role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.Biochim. Biophys. Acta. 2004; 1673: 13-28Google Scholar, 4Chiaradonna F. Ricciardiello F. Palorini R. The nutrient-sensing hexosamine biosynthetic pathway as the hub of cancer metabolic rewiring.Cells. 2018; 7: 53Google Scholar). This role is especially important in the context of disease where metabolic imbalances can be detected via alterations in patterns of glycosylation (5Turner G. N-glycosylation of serum proteins in disease and its investigation using lectins.Clin. Chim. Acta. 1992; 208: 149-171Google Scholar, 6Beisswenger P.J. Makita Z. Curphey T.J. Moore L.L. Jean S. Brinck-Johnsen T. Bucala R. Vlassara H. Formation of immunochemical advanced glycosylation end products precedes and correlates with early manifestations of renal and retinal disease in diabetes.Diabetes. 1995; 44: 824-829Google Scholar, 7Wang J.-Z. Grundke-Iqbal I. Iqbal K. Glycosylation of microtubule–associated protein tau: An abnormal posttranslational modification in Alzheimer's disease.Nat. Med. 1996; 2: 871Google Scholar, 8Dennis J.W. Granovsky M. Warren C.E. Protein glycosylation in development and disease.Bioessays. 1999; 21: 412-421Google Scholar, 9Martin-Rendon E. Blake D.J. Protein glycosylation in disease: New insights into the congenital muscular dystrophies.Trends Pharmacol. Sci. 2003; 24: 178-183Google Scholar, 10Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Google Scholar). Glycan synthesis is nontemplate-directed; therefore, glycan structures are dependent on the availability of their carbohydrate components (i.e., sugars). As a result, glycosylation allows cells to “sense” the availability and abundance of sugars and present and display this information in the form of altered glycan patterns. However, it is not well understood how this information is transduced into functional responses. Galectin 3 is a unique protein that provides a link between sensing changes in glycosylation and modulating cellular functions. Galectin 3 recognizes and binds to carbohydrates and hence can detect changes in glycan patterns (11Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E.G. Yagi F. Kasai K.-i. Oligosaccharide specificity of galectins: A search by frontal affinity chromatography.Biochim. Biophys. Acta. 2002; 1572: 232-254Google Scholar, 12Leffler H. Carlsson S. Hedlund M. Qian Y. Poirier F. Introduction to galectins.Glycoconj. J. 2002; 19: 433-440Google Scholar, 13Nabi I.R. Shankar J. Dennis J.W. The galectin lattice at a glance.J. Cell Sci. 2015; 128: 2213-2219Google Scholar, 14Johannes L. Jacob R. Leffler H. Galectins at a glance.J. Cell Sci. 2018; 131jcs208884Google Scholar). It has also been shown to mediate several cellular functions including clathrin-independent endocytosis (CIE) (15Bresalier R.S. Mazurek N. Sternberg L.R. Byrd J.C. Yunker C.K. Nangia-Makker P. Raz A. Metastasis of human colon cancer is altered by modifying expression of the β-galactoside-binding protein galectin 3.Gastroenterology. 1998; 115: 287-296Google Scholar, 16Furtak V. Hatcher F. Ochieng J. Galectin-3 mediates the endocytosis of β-1 integrins by breast carcinoma cells.Biochem. Biophys. Res. Commun. 2001; 289: 845-850Google Scholar, 17Ochieng J. Furtak V. Lukyanov P. Extracellular functions of galectin-3.Glycoconj. J. 2002; 19: 527-535Google Scholar, 18Sano H. Hsu D.K. Apgar J.R. Yu L. Sharma B.B. Kuwabara I. Izui S. Liu F.-T. Critical role of galectin-3 in phagocytosis by macrophages.J. Clin. Invest. 2003; 112: 389-397Google Scholar, 19Partridge E.A. Le Roy C. Di Guglielmo G.M. Pawling J. Cheung P. Granovsky M. Nabi I.R. Wrana J.L. Dennis J.W. Regulation of cytokine receptors by golgi N-glycan processing and endocytosis.Science. 2004; 306: 120-124Google Scholar, 20Lajoie P. Partridge E.A. Guay G. Goetz J.G. Pawling J. Lagana A. Joshi B. Dennis J.W. Nabi I.R. Plasma membrane domain organization regulates EGFR signaling in tumor cells.J. Cell Biol. 2007; 179: 341-356Google Scholar, 21Lakshminarayan R. Wunder C. Becken U. Howes M.T. Benzing C. Arumugam S. Sales S. Ariotti N. Chambon V. Lamaze C. Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers.Nat. Cell Biol. 2014; 16: 595Google Scholar, 22Mathew M.P. Tan E. Saeui C.T. Bovonratwet P. Sklar S. Bhattacharya R. Yarema K.J. Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells.Oncotarget. 2016; 7: 66491-66511Google Scholar, 23Mathew M.P. Donaldson J.G. Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.J. Biol. Chem. 2018; 293: Scholar, M.P. Donaldson J.G. Glycosylation and glycan interactions can as extracellular clathrin-independent Scholar, M.P. Tan E. Saeui C.T. Bovonratwet P. Liu L. Bhattacharya R. Yarema K.J. Metabolic pancreatic cancer cells to and Med. Chem. 2015; Scholar). The of galectin 3 at the of glycan and modulating cellular function an important role for galectin 3 as a functional in nutrient Galectin 3 is a that is in the and secreted by secretion H. Carlsson S. Hedlund M. Qian Y. Poirier F. Introduction to galectins.Glycoconj. J. 2002; 19: 433-440Google Scholar, of the galectin of Biophys. Acta. 1999; Scholar, B. Plasma membrane and of galectin 3 the of cells Cell Sci. Scholar, in the of galectin-3 for secretion by a pathway the J. 1999; Scholar). Galectin 3 binds to terminal galactose residues and is to detect changes in cell surface glycosylation patterns in which cells detect and present nutrient galectin 3 is unique the galectin of proteins to its to into H. Carlsson S. Hedlund M. Qian Y. Poirier F. Introduction to galectins.Glycoconj. J. 2002; 19: 433-440Google Scholar, 13Nabi I.R. Shankar J. Dennis J.W. The galectin lattice at a glance.J. Cell Sci. 2015; 128: 2213-2219Google Scholar, 14Johannes L. Jacob R. Leffler H. Galectins at a glance.J. Cell Sci. 2018; 131jcs208884Google Scholar). It has also been shown to drive a of cellular functions including and cell (15Bresalier R.S. Mazurek N. Sternberg L.R. Byrd J.C. Yunker C.K. Nangia-Makker P. Raz A. Metastasis of human colon cancer is altered by modifying expression of the β-galactoside-binding protein galectin 3.Gastroenterology. 1998; 115: 287-296Google Scholar, 16Furtak V. Hatcher F. Ochieng J. Galectin-3 mediates the endocytosis of β-1 integrins by breast carcinoma cells.Biochem. Biophys. Res. Commun. 2001; 289: 845-850Google Scholar, 17Ochieng J. Furtak V. Lukyanov P. Extracellular functions of galectin-3.Glycoconj. J. 2002; 19: 527-535Google Scholar, 21Lakshminarayan R. Wunder C. Becken U. Howes M.T. Benzing C. Arumugam S. Sales S. Ariotti N. Chambon V. Lamaze C. Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers.Nat. Cell Biol. 2014; 16: 595Google Scholar, 22Mathew M.P. Tan E. Saeui C.T. Bovonratwet P. Sklar S. Bhattacharya R. Yarema K.J. Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells.Oncotarget. 2016; 7: 66491-66511Google Scholar, 23Mathew M.P. Donaldson J.G. Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.J. Biol. Chem. 2018; 293: Scholar, Y. T. Raz A. Galectin-3 and J. 2002; 19: Scholar, C. C. R. R. A. B. C. G. S. Galectin-3 is essential for cell and and in 2018; Scholar). These galectin 3 as a link between nutrient sensing information and functional A of this function of galectin 3 is that it is an extracellular function and hence there is little known about how this protein the membrane of the galectin of Biophys. Acta. 1999; Scholar, K. secretion of and Cell Biol. 2018; Scholar). via has been as a of its secretion S. J. M.T. T. R. Jacob R. to galectin-3 into for Sci. U. S. A. 2018; 115: a for galectin 3 secretory that secreted galectin 3 and not that not the secretory pathway N. A. P.J. K. A the role of glycosylation in galectin-3 to the cell Cell Sci. Scholar). galectin 3 is in the cytoplasm, it be to O-GlcNAcylation and with O-GlcNAcylation is a posttranslational modification of cytoplasmic It the addition of a N-acetylglucosamine a of a are in this O-GlcNAc which a to a protein and which the O-GlcNAc modification G.W. C. G. between O-GlcNAcylation and in and Scholar, S. Hart G.W. Nutrient of and cell by 2014; Scholar, G.W. of on nutrient sensor that regulates and cellular 2014; Scholar, A little a The cell of Cell Biol. 2015; 208: Scholar). This modification has been observed on proteins and has been shown to modulate protein signaling and in that are to G.W. C. G. between O-GlcNAcylation and in and Scholar, S. Hart G.W. Nutrient of and cell by 2014; Scholar, G.W. of on nutrient sensor that regulates and cellular 2014; Scholar, A little a The cell of Cell Biol. 2015; 208: Scholar). Importantly, the for O-GlcNAcylation is not dependent on is also by cellular and levels. O-GlcNAcylation plays an important role in nutrient sensing (1Wells L. Vosseller K. Hart G. A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance.Cell. Mol. Life Sci. 2003; 60: 222-228Google Scholar, 2Wellen K.E. Thompson C.B. Cellular metabolic stress: Considering how cells respond to nutrient excess.Mol. Cell. 2010; 40: 323-332Google Scholar, S. Hart G.W. Nutrient of and cell by 2014; Scholar, G.W. of on nutrient sensor that regulates and cellular 2014; Scholar). This to galectin 3 changes in cellular O-GlcNAc and these nutrient changes to the extracellular functional we that cellular O-GlcNAc galectin 3 functional changes in CIE. that galectin 3 can as a between nutrient-sensing information by cell surface glycosylation and information these and transducing it into functional biological we to changes in O-GlcNAcylation the of galectin 3 to with cell surface A has that O-GlcNAcylation to secretion of galectins R. The role of galectins and O-GlcNAc in of cellular Scholar). we the secretion of galectin 3 with altered the of O-GlcNAcylation on galectin 3 we to a of C. a for in metabolic Biol. Chem. 2015; Scholar). of the of O-GlcNAc residues and to an in O-GlcNAcylation levels. an to the of galectin 3 secreted cell the of a we that in and there a significant in galectin 3 secretion This in secretion also observed in we in a cancer cell as glycosylation changes have been to be of cancer L. Jacob R. Leffler H. Galectins at a glance.J. Cell Sci. 2018; 131jcs208884Google Scholar, J. D.J. of glycosylation in 2016; 7: Scholar, A. M. R. L.L. Protein glycosylation and tumor alterations cancer Scholar, O-GlcNAc in An 2018; Scholar). We to in cells and in a of and a significant in O-GlcNAc and to the of these proteins M. M.P. D.J. J. M. K. S. C.E. L. An with in O-GlcNAc J. Scholar, K.E. A regulates O-GlcNAc and O-GlcNAc Scholar). in significant in O-GlcNAcylation and Galectin 3 secretion a using an In these we observed a significant of galectin 3 secretion with we observed in the changes in galectin 3 by we to O-GlcNAc and to of and and to of O-GlcNAcylation and Galectin 3 secretion the of by with These results indicate that of the secretion of galectin 3. these that O-GlcNAcylation galectin 3 O-GlcNAcylation galectin 3 secretion by a A is that O-GlcNAcylation of galectin 3 signals its the cell, and its is to it the It is that not secretion it not the cells were in a which be in on the cytoplasmic synthesis of galectin 3 and its we that galectin 3 be by O-GlcNAcylation with galectin 3 its are we a by of cytoplasmic and binds terminal and we that galectin 3 can be with In to galectin 3 can be we an in O-GlcNAcylation we galectin 3 with and we the with that the on a and detected with The galectin 3 in to the In an to how O-GlcNAc with galectin we for galectin 3 using a and to This galectin 3 with several proteins in these that galectin 3 can be by and that it with several that galectin 3 can be O-GlcNAc we to how this modification this we a galectin 3 in to detect galectin 3 and secretion in galectin the cellular and cytoplasmic of proteins using the which of an to which can be to Western of the proteins an with the galectin in the cellular and This that galectin is the in the secreted were to be detected by Western we were to detect the of secreted galectin there little to the secreted to the of galectin 3 that there a significant in O-GlcNAcylation in the secreted of galectin 3 These results indicate that the O-GlcNAcylation of galectin 3 play a role in its secretion and that the of O-GlcNAcylation is an important galectin 3 can be we the protein of galectin 3 for predicted of O-GlcNAcylation using the were of O-GlcNAcylation predicted with a of of these predicted in the of galectin 3 the is to the In to the role of O-GlcNAcylation on galectin how changes in O-GlcNAcylation of galectin 3 its we a of galectin with of predicted O-GlcNAcylation These were in and a the of galectin secreted using a the in which in the were were secreted at a galectin The the not to secretion on its we that not secretion of the in which in the were in addition to a at as it the These that O-GlcNAcylation the of galectin 3 plays a role in its these results that galectin 3 to be and in to be there a link between nutrient and galectin 3 cells were in with in and the cells secreted galectin 3 and cells in Western showed that O-GlcNAcylation with cellular galectin 3 were and The cells were in with and with by there a significant of the nutrient in galectin 3 secretion in the This that at a of the in galectin 3 secretion by dynamic O-GlcNAcylation. Western that O-GlcNAcylation with and to an in O-GlcNAcylation cellular galectin 3 were and not we also the and We that these cells have a dramatic difference between and In the there a significant in galectin 3 secretion in cells in there significant in galectin 3 secretion in in as with This is with a in which dynamic O-GlcNAcylation to changes in galectin 3 that O-GlcNAcylation galectin 3 However, it this known extracellular functions of galectin 3 CIE. In to the of O-GlcNAc on galectin 3 we the of We observed an in The of galectin 3 secretion with an in the that O-GlcNAc changes the availability of extracellular galectin 3. we endocytosis of which has been shown to be to changes in interactions M.P. Donaldson J.G. Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.J. Biol. Chem. 2018; 293: as a As of by changes in O-GlcNAcylation cells their and that information to modulate an essential we how the galectin is to status O-GlcNAcylation and that information interactions with extracellular glycans to show that secreted galectin 3 is and that in O-GlcNAc to altered patterns of galectin 3 show significant galectin 3 secretion and and are to galectin 3 secretion in cells These a link between O-GlcNAcylation and nonclassical such as and galectins are also secreted by nonclassical mechanisms and to their cytoplasmic synthesis also have their secretion via O-GlcNAcylation. that galectin 3 can be by and that modification in the of galectin 3 secretion and investigation into the O-GlcNAc status of secreted proteins such as and galectins is in to O-GlcNAc plays a role in modulating the nonclassical secretory this has on the of of O-GlcNAc on galectin 3 it that there are as interactions with proteins and posttranslational Importantly, we that changes in nutrient status of the cell galectin 3 secretion and that this is O-GlcNAcylation These that the hexosamine biosynthetic pathway can to extracellular glycosylation of galectin 3. indicate that a complex and nuanced role galectin 3 plays as a of nutrient sensing to to the in this we show that the essential function of is by changes in have shown that interactions be a by which is M.P. Tan E. Saeui C.T. Bovonratwet P. Sklar S. Bhattacharya R. Yarema K.J. Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells.Oncotarget. 2016; 7: 66491-66511Google Scholar, 23Mathew M.P. Donaldson J.G. Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.J. Biol. Chem. 2018; 293: Scholar, M.P. Tan E. Saeui C.T. Bovonratwet P. Liu L. Bhattacharya R. Yarema K.J. Metabolic pancreatic cancer cells to and Med. Chem. 2015; Scholar). We show that of O-GlcNAcylation by of and to a in galectin 3 secretion and that the in extracellular galectin 3 to an in This of with the observed galectin 3 in cells M.P. Donaldson J.G. Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.J. Biol. Chem. 2018; 293: Scholar). the role of O-GlcNAcylation and nutrient sensing in galectin 3 secretion and by O-GlcNAcylation to of of we that be we that with the on secretion as the of O-GlcNAc of galectin 3 and investigation be to the by which O-GlcNAc regulates galectin 3 it is that there is an of with protein availability a In addition to its unique biological galectin 3 has also been observed in a of disease cancer to diabetes and disease I. N. C. E. S. of galectin-3 in the of and cancer Res. Scholar, M. Oka N. R. K. T. of galectin-3 in human Med. Invest. Scholar, M. A. P. and of galectin-3 in with to and of with in the J. Scholar, R.S. galectin-3 precedes the development of 24: Scholar, Y. M. K. H. G. R. R. H. regulates and Sci. 2016; Scholar, J. galectin-3 for the of A J. 2016; Scholar, K. K. Y. not are in for the role of Scholar, J. M. M. in diabetes with of 2018; 7: Scholar). the molecular mechanisms by interactions and how galectin 3 secretion is with insights into how galectins and glycosylation drive the of several and how the can disease progression. and cells were in with serum and were at in a a of T. in and a of in and the cells were in the for to the of the a of in and the cells were in for to the of the were A galectin 3 A that galectin 3 A that Cell A that and were also An 3 and to and were the and cells on were on and of at of for were on at for to were for of that at in and to the and the of that at as well as the that on for using a of which these of were in The cells were for using a of in The were at for with and cell in The were in and a with and were on were at using a with a and and each were with with the each the at cells were for each were with which are on the and such that the the dynamic The to the of were into the An for each using its The and were and the integrated for the The cell and the The integrated for the to the for each The is with the the were to the were on a at a of 3 in of in with and a the (i.e., were and at for to cell were for galectin 3 using a human galectin 3 as by the in the well were to and Galectin 3 in the to cell were in and with a of human human using as by the were in to cells were by Western the cells were in by using a cell and by at for were in protein were and using a were by to to and with the and 3 were using Protein were using the were using for and cytoplasmic were using the and cytoplasmic of in of to and at The the were in for were the using in for on and were were as described for Western galectin 3 using the and to the and of these were using the 3 3 3 and using the 3 the for 3 were 3 using the 3 the for were 3 and using the 3 the for were and were in a cells were with galectin 3 of at of well using as by the were to The by of 3 of galectin of and the to with were by galectin 3 the A by for galectin 3 in the The were for in a The were to and at for The were with of and at for the the were and at for 3 of and for at The were for in and proteins were by to to in for and with and 3 were using a The galectin 3 on the to of R. of glycosylation in to protein at Scholar, R. S. of glycosylation the human and the to protein 2002; Scholar). The a of predicted with of a with cells were with and in of with were at for were to The were using 3 protein The were for on with in were with and for in Galectin 3 the were on and with protein were with in with at for and with in the at for and were with of at to the The were and in and by to the The were and with and for The were by on an to a proteins were a 3 and on an at a and with A were with a of to at a of The in to between and to by and to were using using H. as the and were were with a of and a of of of residues of of residues of of and and to and dynamic were were by with the of The have been to the via the with a with galectin cells were with and in of with The also in and at for to cell were to Cell were at for were to The were using of of an to to The were for on with in were with and for in and proteins of and of the were by to to and with the 3 and were using a were on a at a of in of in were with galectin 3 a of the with of with serum and the were and at for to cell and of the to a in The cells were also by with for at were and in of of the cell also to a in in each well using a the and cell the the to the in the cell biological were for each and were as In with determined using a with a to of with the a to of A with there were In with conditions, an to The in where were The have been to the via the with are the This The that have of with the of this The were by and at the and by This by of and by of The is the of the and not the of the of M. P. M. M. P. M. and L. K. A. J. A. H. M. P. M. and L. K. A. M. P. M. J. A. H. L. K. A. M. P. M. and L. K. A. J. G. and J. A. H. and with with with with

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