A Mutation in Aminopeptidase N (CD13) Isolated from a Patient Suffering from Leukemia Leads to an Arrest in the Endoplasmic Reticulum

Abstract

Human aminopeptidase N (APN) is used as a routine marker for myelomonocytic cells in hematopoietic malignant disorders. Its gene and surface expressions are increased in cases of malignant transformation, inflammation, or T cell activation, whereas normal B and resting T cells lack detectable APN protein expression. In this study we elucidated the intracellular distribution, expression pattern, and enzymatic activity of a naturally occurring mutation in the coding region of the APN gene. At physiological temperatures the mutant protein is enzymatically inactive, persists as a mannose-rich polypeptide in the endoplasmic reticulum, and is ultimately degraded by an endoplasmic reticulum-associated degradation pathway. It shows in part the distinct behavior of a temperature-sensitive mutant with a permissive temperature of 32 °C, leading to correct sorting of the Golgi compartment accompanied by the acquisition of proper glycosylation but without reaching the cell-surface membrane and without regaining its enzymatic activity. Because the patient bearing this mutation suffered from leukemia, possible links to the pathogenesis of leukemia are discussed. Human aminopeptidase N (APN) is used as a routine marker for myelomonocytic cells in hematopoietic malignant disorders. Its gene and surface expressions are increased in cases of malignant transformation, inflammation, or T cell activation, whereas normal B and resting T cells lack detectable APN protein expression. In this study we elucidated the intracellular distribution, expression pattern, and enzymatic activity of a naturally occurring mutation in the coding region of the APN gene. At physiological temperatures the mutant protein is enzymatically inactive, persists as a mannose-rich polypeptide in the endoplasmic reticulum, and is ultimately degraded by an endoplasmic reticulum-associated degradation pathway. It shows in part the distinct behavior of a temperature-sensitive mutant with a permissive temperature of 32 °C, leading to correct sorting of the Golgi compartment accompanied by the acquisition of proper glycosylation but without reaching the cell-surface membrane and without regaining its enzymatic activity. Because the patient bearing this mutation suffered from leukemia, possible links to the pathogenesis of leukemia are discussed. Membrane alanyl aminopeptidase (aminopeptidase N, APN 2The abbreviations used are: APN, aminopeptidase N; GFP, green fluorescent protein; CD, cluster of differentiation; GT, galactosyltransferase; ER, endoplasmic reticulum; mAb, monoclonal antibody; endo H, endo-β-N-acetylglucosaminidase H; TEMED, N,N,N′,N′-tetramethylethylenediamine; PBS, phosphate-buffered saline. 2The abbreviations used are: APN, aminopeptidase N; GFP, green fluorescent protein; CD, cluster of differentiation; GT, galactosyltransferase; ER, endoplasmic reticulum; mAb, monoclonal antibody; endo H, endo-β-N-acetylglucosaminidase H; TEMED, N,N,N′,N′-tetramethylethylenediamine; PBS, phosphate-buffered saline. (1Barnes K. is a protein is the surface of a of cell in and (1Barnes K. of its are is the from the N of leading to degradation of K. K. K. K. K. K. and its APN is in degradation of in the and of of and in and APN to degradation and of and are of its distinct as a for the and or K. K. of APN to the of cells in a K. K. as as and T cell leukemia cells in K. K. K. APN is of by in the and of T cells and T cell K. K. K. of malignant and cell in In T and B cell to of APN K. K. is a of APN expression to or from malignant of study in the gene coding for APN in and of cases of leukemia and K. and of mutant from a patient with the of a mutation to protein and and the of a of the mutation APN expression and enzymatic and and from and for from from and protein from and from and from and from by from with and from from of APN, with GFP, the from of APN from cells the in the expression to the of the with the in from to the by the as the and mutation by of and cells with by as and used the the cells cells in of for the and in with for of the cells with phosphate-buffered and with of and with and for of with of protein the with the and of protein as by to the and by a In of in the and degradation of the mutant the cells for with and for to in the or of an of the used a of cells as and the protein in the as as the of used in the of the mutant to the and mutant APN from the surface of cells as cells with of for °C, °C, or 32 the cells to °C, and or mutant APN the cell surface by to the for cells in and with protein to of from cells with or for by and of of of cells a with a and by with the of an or the of a and the for or as of APN in and by for for and the in of the and used to for APN activity. activity of by the of K. to of and cell from a of a cell the of of It APN or cells with or by as of cells with or in to cells by as a as cells for in the the membrane and to the of the and the cell a of cells of a patient from and as K. by and a mutation an from T to to of by of T in the the of this mutation mutant and a of as an endo mannose-rich polypeptide whereas the shows the of a of the mannose-rich and a of the of APN by of with endo to a of the mannose-rich to in whereas the the shows in a patient from K. and is for is as an endo mannose-rich and endo of APN cells with for of with with or without endo for to by of mutant and APN mutant as a mannose-rich the the glycosylation and endo of we the mutant protein is to in the Golgi to a protein of and intracellular in the of APN cells with the of or cells with for and with for the of with and with endo or to by this used to the of mutant protein a distinct of with the In in Golgi as as the cell-surface of to APN its normal its as by enzymatic of the APN with an or a Golgi cells with and a the of with and with the of a marker for the ER, and an of the cells the intracellular we and the mutant with the of a marker for the ER, and a or a the of with in APN with the and Golgi and the cell-surface whereas the protein a to the marker with the the intracellular of the mutant in the as a mannose-rich we to the and the of this a in the mutant to the and detectable of in its the the mutant is and its is of the the possible degradation of by an degradation and we the mutant and its in a an of the of the mutant protein the with the in a in the of the mutant is in the of the of the mutant degradation of as with without the and of as with without the is with a degradation of the cells with the of or cells with for and with for the in the or of of with and with endo or to by mutant of membrane in the of temperature-sensitive the permissive temperature and are to the to the Golgi and ultimately to the cell surface K. At the degradation in mannose-rich the is we its cells or 32 for to and In the of the and in the glycosylation of the mutant protein whereas 32 a glycosylation pattern, this temperature the protein a with a by a of to 32 glycosylation is to the in to the the mannose-rich of but In to to the this to protein of and mutant APN 32 cells with the of or cells 32 for with for and with for the of with to by we is to the cell-surface membrane by a cell-surface 32 of detectable the cell surface 32 °C, whereas in the of protein the cell membrane 32 or a of the 32 In the the mannose-rich as as the the mutant is to the Golgi but the cell are by of the of in cells 32 by In cells the mutant protein in distinct with the as by with the cell-surface membrane APN in cells in the of the mutant protein to the Golgi used as a as 32 as surface of and cells with of and for 32 °C, and in the of with or without an of the cells to °C, and or mutant APN the cell surface by to the for cell and from the the cell surface of with 32 cells with and as Golgi the cells for 32 and by of protein enzymatic activity is enzymatically 32 as as physiological whereas a activity temperatures we the protein 32 °C, is of reaching the cell-surface membrane of regaining enzymatic activity. with the of or cells in and by for for and the in of PBS, and the activity of APN as APN activity as and cells used as a of temperature a to 32 we the of the APN protein with degraded by of and increased of a this In degraded the of the APN mutant to the of in an In the of the the cells 32 for to a 32 with physiological temperature cells with the of or and with for of with to a for the to by from the we the cell with and and and a for with in an expression the cell surface of APN, whereas the mutant of in cells increased with In cells with in of cells increased with but with expression of and in the cell shows the surface whereas is of and of with cells In expression of or of or mutation in a patient from leukemia in the intracellular and of the membrane of the mutant protein to its in the as an protein is ultimately degraded by an degradation pathway. of in the is to to or of a temperature-sensitive In a permissive temperature of 32 in from the to the Golgi and acquisition of enzymatic of for and the APN by of a to the of the APN protein are in a protein the to from the the Golgi physiological is to a is we its the mutant a in the protein with a of a temperature of 32 in a but in a in as from the we the is a of temperatures a in the APN of are and with a of the temperature but the mutant is to the naturally occurring mutation protein and in the APN gene. from a patient from leukemia, is to or to the cells of and the cluster of the surface as in K. Because of or malignant transformation, B and T cells APN gene and surface expression. expression of APN malignant the of APN surface expression and T and the of aminopeptidase to a of APN expression to K. K. K. is by the in the gene coding for APN in a of from leukemia and are or by of APN to the malignant to this is to the a mutation enzymatic and we the in a patient from and and the of the mutation is with APN the of APN to or APN is in and of cells and we the and of malignant of or a cell of expression this cell from hematopoietic cell for APN expression K. K. increased expression of is with and are in with in to an of APN enzymatic activity K. is of APN to a malignant the and of cells a of and from APN in intracellular in the of a expression are the in or of APN, in to the lack of enzymatic the of a cell to malignant the to in as as we the mutant the a mutation in the coding for APN and Membrane alanyl aminopeptidase (aminopeptidase N, APN 2The abbreviations used are: APN, aminopeptidase N; GFP, green fluorescent protein; CD, cluster of differentiation; GT, galactosyltransferase; ER, endoplasmic reticulum; mAb, monoclonal antibody; endo H, endo-β-N-acetylglucosaminidase H; TEMED, N,N,N′,N′-tetramethylethylenediamine; PBS, phosphate-buffered saline. 2The abbreviations used are: APN, aminopeptidase N; GFP, green fluorescent protein; CD, cluster of differentiation; GT, galactosyltransferase; ER, endoplasmic reticulum; mAb, monoclonal antibody; endo H, endo-β-N-acetylglucosaminidase H; TEMED, N,N,N′,N′-tetramethylethylenediamine; PBS, phosphate-buffered saline. (1Barnes K. is a protein is the surface of a of cell in and (1Barnes K. of its are is the from the N of leading to degradation of K. K. K. K. K. K. and its APN is in degradation of in the and of of and in and APN to degradation and of and are of its distinct as a for the and or K. K. of APN to the of cells in a K. K. as as and T cell leukemia cells in K. K. K. APN is of by APN in the and of T cells and T cell K. K. K. of malignant and cell in In T and B cell to of APN K. K. is a of APN expression to or from malignant of study in the gene coding for APN in and of cases of leukemia and K. and of mutant from a patient with the of a mutation to protein and and the of a of the mutation APN expression and enzymatic activity. and and from and for from from and protein from and from and from and from by from with and from from of APN, with GFP, the from of APN from cells the in the expression to the of the with the in from to the by the as the and mutation by of and cells with by as and used the the cells cells in of for the and in with for of the cells with phosphate-buffered and with of and with and for of with of protein the with the and of protein as by to the and by a In of in the and degradation of the mutant the cells for with and for to in the or of an of the used a of cells as and the protein in the as as the of used in the of the mutant to the and mutant APN from the surface of cells as cells with of for °C, °C, or 32 the cells to °C, and or mutant APN the cell surface by to the for cells in and with protein to of from cells with or for by and of of of cells a with a and by with the of an or the of a and the for or as of APN in and by for for and the in of the and used to for APN activity. activity of by the of K. to of and cell from a of a cell the of of It APN or cells with or by as of cells with or in to cells by as a as cells for in the the membrane and to the of the and the cell a and and from and for from from and protein from and from and from and from by from with and from from of APN, with GFP, the from of APN from cells the in the expression to the of the with the in from to the by the as the and mutation by of and cells with by as and used the the cells cells in of for the and in with for of the cells with phosphate-buffered and with of and with and for of with of protein the with the and of protein as by to the and by a In of in the and degradation of the mutant the cells for with and for to in the or of an of the used a of cells as and the protein in the as as the of used in the of the mutant to the and mutant APN from the surface of cells as cells with of for °C, °C, or 32 the cells to °C, and or mutant APN the cell surface by to the for cells in and with protein to of from cells with or for by and of of of cells a with a and by with the of an or the of a and the for or as of APN in and by for for and the in of the and used to for APN activity. activity of by the of K. to of and cell from a of a cell the of of It APN or cells with or by as of cells with or in to cells by as a as cells for in the the membrane and to the of the and the cell a of cells of a patient from and as K. by and a mutation an from T to to of by of T in the the of this mutation mutant and a of as an endo mannose-rich polypeptide whereas the shows the of a of the mannose-rich and a of the of APN by of with endo to a of the mannose-rich to in whereas the the shows in a patient from K. and is for is as an endo mannose-rich of mutant and APN mutant as a mannose-rich the the glycosylation and endo of we the mutant protein is to in the Golgi to a protein of and intracellular in the of APN cells with the of or cells with for and with for the of with and with endo or to by this used to the of mutant protein a distinct of with the In in Golgi as as the cell-surface of to APN its normal its as by enzymatic of the APN with an or a Golgi cells with and a the of with and with the of a marker for the ER, and an of the cells the intracellular we and the mutant with the of a marker for the ER, and a or a the of with in APN with the and Golgi and the cell-surface whereas the protein a to the marker with the the intracellular of the mutant in the as a mannose-rich we to the and the of this a in the mutant to the and detectable of in its the the mutant is and its is of the the possible degradation of by an degradation and we the mutant and its in a an of the of the mutant protein the with the in a in the of the mutant is in the of the of the mutant degradation of as with without the and of as with without the is with a degradation of the cells with the of or cells with for and with for the in the or of of with and with endo or to by mutant of membrane in the of temperature-sensitive the permissive temperature and are to the to the Golgi and ultimately to the cell surface K. At the degradation in mannose-rich the is we its cells or 32 for to and In the of the and in the glycosylation of the mutant protein whereas 32 a glycosylation pattern, this temperature the protein a with a by a of to 32 glycosylation is to the in to the the mannose-rich of but In to to the this to protein of and mutant APN 32 cells with the of or cells 32 for with for and with for the of with to by we is to the cell-surface membrane by a cell-surface 32 of detectable the cell surface 32 °C, whereas in the of protein the cell membrane 32 or a of the 32 In the the mannose-rich as as the the mutant is to the Golgi but the cell are by of the of in cells 32 by In cells the mutant protein in distinct with the as by with the cell-surface membrane APN in cells in the of the mutant protein to the Golgi used as a as 32 as surface of and cells with of and for 32 °C, and in the of with or without an of the cells to °C, and or mutant APN the cell surface by to the for cell and from the the cell surface of with 32 cells with and as Golgi the cells for 32 and by of protein enzymatic activity is enzymatically 32 as as physiological whereas a activity temperatures we the protein 32 °C, is of reaching the cell-surface membrane of regaining enzymatic activity. with the of or cells in and by for for and the in of PBS, and the activity of APN as APN activity as and cells used as a of temperature a to 32 we the of the APN protein with degraded by of and increased of a this In degraded the of the APN mutant to the of in an In the of the the cells 32 for to a 32 with physiological temperature cells with the of or and with for of with to a for the to by from the we the cell with and and and a for with in an expression the cell surface of APN, whereas the mutant of in cells increased with In cells with in of cells increased with but with expression of and in the cell shows the surface whereas is of and of with cells In expression of or of or of cells of a patient from and as K. by and a mutation an from T to to of by of T in the the of this mutation mutant and a of as an endo mannose-rich polypeptide whereas the shows the of a of the mannose-rich and a of the of APN by of with endo to a of the mannose-rich to in whereas the the shows in a patient from K. and is for is as an endo mannose-rich of mutant and APN mutant as a mannose-rich the the glycosylation and endo of we the mutant protein is to in the Golgi to a protein of and intracellular in the this used to the of mutant protein a distinct of with the In in Golgi as as the cell-surface of to APN its normal its as by enzymatic the intracellular we and the mutant with the of a marker for the ER, and a or a the of with in APN with the and Golgi and the cell-surface whereas the protein a to the marker with the the intracellular of the mutant in the as a mannose-rich we to the and the of this a in the mutant to the and detectable of in its the the mutant is and its is of the the possible degradation of by an degradation and we the mutant and its in a an of the of the mutant protein the with the in a in the of the mutant is in the of the of the mutant degradation of as with without the and of as with without the is with a degradation of the mutant of membrane in the of temperature-sensitive the permissive temperature and are to the to the Golgi and ultimately to the cell surface K. At the degradation in mannose-rich the is we its cells or 32 for to and In the of the and in the glycosylation of the mutant protein whereas 32 a glycosylation pattern, this temperature the protein a with a by a of to 32 glycosylation is to the in to the the mannose-rich of but In to to the this to protein we is to the cell-surface membrane by a cell-surface 32 of detectable the cell surface 32 °C, whereas in the of protein the cell membrane 32 or a of the 32 In the the mannose-rich as as the the mutant is to the Golgi but the cell are by of the of in cells 32 by In cells the mutant protein in distinct with the as by with the cell-surface membrane APN in cells in the of the mutant protein to the Golgi used as a as 32 as of protein enzymatic activity is enzymatically 32 as as physiological whereas a activity temperatures we the protein 32 °C, is of reaching the cell-surface membrane of regaining enzymatic activity. we the of the APN protein with degraded by of and increased of a this In degraded the of the APN mutant to the of in an In the of the the cells 32 for to a 32 with physiological temperature from the we the cell with and and and a for with in an expression the cell surface of APN, whereas the mutant of in cells increased with In cells with in of cells increased with but with mutation in a patient from leukemia in the intracellular and of the membrane of the mutant protein to its in the as an protein is ultimately degraded by an degradation pathway. of in the is to to or of a temperature-sensitive In a permissive temperature of 32 in from the to the Golgi and acquisition of enzymatic of for and the APN by of a to the of the APN protein are in a protein the to from the the Golgi physiological is to a is we its the mutant a in the protein with a of a temperature of 32 in a but in a in as from the we the is a of temperatures a in the APN of are and with a of the temperature but the mutant is to the naturally occurring mutation protein and in the APN gene. from a patient from leukemia, is to or to the cells of and the cluster of the surface as in K. Because of or malignant transformation, B and T cells APN gene and surface expression. expression of APN malignant the of APN surface expression and T and the of aminopeptidase to a of APN expression to K. K. K. is by the in the gene coding for APN in a of from leukemia and are or by of APN to the malignant to this is to the a mutation enzymatic and we the in a patient from and and the of the mutation is with APN the of APN to or APN is in and of cells and we the and of malignant of or a cell of expression this cell from hematopoietic cell for APN expression K. K. increased expression of is with and are in with in to an of APN enzymatic activity K. is of APN to a malignant the and of cells a of and from APN in intracellular in the of a expression are the in or of APN, in to the lack of enzymatic the of a cell to malignant the to in as as we the mutant the a mutation in the coding for APN and mutation in a patient from leukemia in the intracellular and of the membrane of the mutant protein to its in the as an protein is ultimately degraded by an degradation pathway. of in the is to to or of a temperature-sensitive In a permissive temperature of 32 in from the to the Golgi and acquisition of enzymatic of for and the APN by of a to the of the APN protein are in a protein the to from the the Golgi physiological is to a is we its the mutant a in the protein with a of a temperature of 32 in a but in a in as from the we the is a of temperatures a in the APN of are and with a of the temperature but the mutant is to the naturally occurring mutation protein and in the APN gene. from a patient from leukemia, is to or to the cells of and the cluster of the surface as in K. Because of or malignant transformation, B and T cells APN gene and surface expression. expression of APN malignant the of APN surface expression and T and the of aminopeptidase to a of APN expression to K. K. K. is by the in the gene coding for APN in a of from leukemia and are or by of APN to the malignant to this is to the a mutation enzymatic and we the in a patient from and and the of the mutation is with APN the of APN to or APN is in and of cells and we the and of malignant of or a cell of expression this cell from hematopoietic cell for APN expression K. K. increased expression of is with and are in with in to an of APN enzymatic activity K. is of APN to a malignant the and of cells a of and from APN in intracellular in the of a expression are the In in or of APN, in to the lack of enzymatic the of a cell to malignant the to in as as we the mutant the a mutation in the coding for APN and