Accumulation of Pyrraline-modified Albumin in Phagocytes due to Reduced Degradation by Lysosomal Enzymes

Abstract

Previous studies suggested that the interaction between proteins modified by advanced glycation end products (AGEs) and cells, such as macrophages, may be involved in diabetic angiopathy. Pyrraline is one of the AGEs and known to be elevated in plasma of diabetic rats and humans, and is present in vascular lesions of diabetic and elderly subjects. We examined whether modification of albumin by pyrraline influences its degradation by macrophage-like cell line, P388D1 cells. Degradation of pyrraline-modified albumin by these cells was diminished, causing accumulation of the albumin in these cells. The susceptibility of pyrraline-modified albumin to lysosomal proteolytic enzymes was reduced by approximately 40% in vitro, while lysosomal activity in the cells per se was not affected. This phenomenon was also observed when human monocytes were used instead of P388D1 cells. Our results suggest that accumulation of pyrraline-modified albumin in P388D1 cells is due to the reduced susceptibility of the protein to lysosomal enzymatic degradation. Such alterations in the interaction between AGEs-modified protein and phagocytes may contribute to angiopathy in elderly subjects and patients with diabetes. Previous studies suggested that the interaction between proteins modified by advanced glycation end products (AGEs) and cells, such as macrophages, may be involved in diabetic angiopathy. Pyrraline is one of the AGEs and known to be elevated in plasma of diabetic rats and humans, and is present in vascular lesions of diabetic and elderly subjects. We examined whether modification of albumin by pyrraline influences its degradation by macrophage-like cell line, P388D1 cells. Degradation of pyrraline-modified albumin by these cells was diminished, causing accumulation of the albumin in these cells. The susceptibility of pyrraline-modified albumin to lysosomal proteolytic enzymes was reduced by approximately 40% in vitro, while lysosomal activity in the cells per se was not affected. This phenomenon was also observed when human monocytes were used instead of P388D1 cells. Our results suggest that accumulation of pyrraline-modified albumin in P388D1 cells is due to the reduced susceptibility of the protein to lysosomal enzymatic degradation. Such alterations in the interaction between AGEs-modified protein and phagocytes may contribute to angiopathy in elderly subjects and patients with diabetes. INTRODUCTIONMaillard reaction (glycation) is thought to play a role in the pathogenesis of angiopathy in diabetes and aging process (1Cohen M.P. Diabetes and Protein Glycosylation. Springer-Verlag, New York1986Crossref Google Scholar, 2Brownlee M. Cerami A. Vlassara H. N. Engl. J. Med. 1988; 318: 1315-1321Crossref PubMed Scopus (2324) Google Scholar, 3Monnier V.M. Baynes J.W. The Maillard Reaction in Aging Diabetes and Nutrition. Alan R. Liss, New York1989Google Scholar). The advanced stage of this reaction that leads to the formation of advanced glycation end products (AGEs) 1The abbreviations used are: AGEadvanced glycation end productBSAbovine serum albuminPyr-BSApyrraline-modified BSARAGEreceptor for AGEVCAM-1vascular cell adhesion molecule-1PBSphosphate-buffered salineELISAenzyme-linked immunosorbent assay. is very complex due to several possible metabolic pathways. Despite this complexity, the structures of several AGEs have been recently described, such as that of pyrraline (4Njoroge F.G. Sayre L.M. Monnier V.M. Carbohydr. Res. 1987; 167: 211-220Crossref PubMed Scopus (115) Google Scholar), pentosidine (5Sell D.R. Monnier V.M. J. Biol. Chem. 1989; 264: 21597-21602Abstract Full Text PDF PubMed Google Scholar), cross-line (6Nakamura K. Hasegawa T. Fukunaga Y. Ienaga K. Chem. Soc. Chem. Commun. 1992; 14: 992-994Crossref Google Scholar), and pyrropyridine (7Hayase F. Hinuma H. Asano M. Kato H. Arai S. Biosci. Biotechnol. Biochem. 1994; 58: 1936-1937Crossref Google Scholar). Carboxymethyllysine is also formed by oxidation of Amadori products (8Dunn J.A. McCance D.R. Thorpe S.R. Lyons T.J. Baynes J.W. Biochemistry. 1991; 30: 1205-1210Crossref PubMed Scopus (284) Google Scholar). Pyrraline is one of the AGEs derived from the reaction of glucose with the lysine amino group on proteins. Several investigators have demonstrated the preferential accumulation of AGEs in diabetic tissues (9Nakayama H. Mitsuhashi T. Kuwajima S. Aoki S. Kuroda Y. Itoh T. Nakagawa S. Diabetes. 1993; 42: 345-350Crossref PubMed Scopus (134) Google Scholar, 10Mitsuhashi T. Nakayama H. Itoh T. Kuwajima S. Aoki S. Atsumi T. Koike T. Diabetes. 1993; 42: 826-832Crossref PubMed Scopus (95) Google Scholar, 11Nakamura Y. Horii Y. Nishino T. Shiiki H. Sakaguchi Y. Kagoshima T. Dohi K. Makita Z. Vlassara H. Bucala R. Am. J. Pathol. 1993; 143: 1649-1656PubMed Google Scholar, 12Kume S. Takeya M. Mori T. Araki N. Suzuki H. Horiuchi S. Kodama T. Miyauchi Y. Takahashi K. Am. J. Pathol. 1995; 147: 654-667PubMed Google Scholar, 13Makita Z. Vlassara H. Cerami A. Bucala R. J. Biol. Chem. 1992; 267: 5133-5138Abstract Full Text PDF PubMed Google Scholar). Plasma pyrraline levels are also elevated in diabetic rats and humans as determined by ELISA using antibody to pyrraline (14Hayase F. Nagaraj R.H. Miyata S. Njoroge F.G. Monnier V.M. J. Biol. Chem. 1989; 264: 3758-3764Abstract Full Text PDF PubMed Google Scholar, 15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). In addition, Porterootin et al. (16Porterootin M. Nagaraj R.H. Monnier V.M. Biochim. Biophys. Acta. 1995; 1247: 74-80Crossref PubMed Scopus (87) Google Scholar) demonstrated the existence of pyrraline in vivo using high performance liquid chromatography. They also showed that pyrraline in vivo could react with other amino acids on proteins to form cross-links (17Nagaraj R.H. Porterootin M. Monnier V.M. Arch. Biochem. Biophys. 1996; 325: 152-158Crossref PubMed Scopus (52) Google Scholar). It was also demonstrated that pyrraline is found in vascular lesions of diabetic and elderly subjects using immunohistochemical techniques (15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). Among several pathways forming pyrraline, highly reactive dicarbonyl compounds, such as 3-deoxyglucosone, were identified as precursors reacting with free amino groups to form pyrraline (14Hayase F. Nagaraj R.H. Miyata S. Njoroge F.G. Monnier V.M. J. Biol. Chem. 1989; 264: 3758-3764Abstract Full Text PDF PubMed Google Scholar, 15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). We recently reported that plasma 3-deoxyglucosone levels are elevated in diabetic rats using specific high performance liquid chromatography assay (18Yamada H. Miyata S. Igaki N. Yatabe H. Miyauchi Y. Ohara T. Sakai M. Shoda H. Oimomi M. Kasuga M. J. Biol. Chem. 1994; 269: 20275-20280Abstract Full Text PDF PubMed Google Scholar).AGEs are also known to alter the structural and functional properties of proteins. Furthermore, a pathological role of the interaction between AGEs-modified proteins and cells for diabetic complications has been recently proposed. Several cell surface proteins are thought to recognize AGEs (19Vlassara H. Brownlee M. Cerami A. Proc. Natl. Sci. U. S. A. 1985; 82: 5588-5592Crossref PubMed Scopus (287) Google Scholar, 20Schmidt A.M. Vianna M. Gerlach M. Brett J. Ryan J. Kao J. Esposito C. Hegarty H. Hurley W. Clauss M. Wang F. Pan Y-C.E. Tsang T.C. Stern D. J. Biol. Chem. 1992; 267: 14987-14997Abstract Full Text PDF PubMed Google Scholar, 21Araki N. Higashi T. Mori T. Shibayama R. Kawabe Y. Kodama T. Takahashi K. Shichiri M. Horiuchi S. Eur. J. Biochem. 1995; 230: 408-415Crossref PubMed Scopus (223) Google Scholar). Furthermore, interaction between AGEs and these proteins induces a variety of secondary effects. Vlassara et al. (22Vlassara H. Brownlee M. Manogue K.R. Dinarello C. Pasagian A. Science. 1988; 240: 1546-1548Crossref PubMed Scopus (530) Google Scholar) demonstrated that macrophages secrete cytokines, such as tumor necrosis factor and interleukin-1, following the recognition of AGEs through specific receptors. They also showed the induction of insulin-like growth factor in human monocytes by AGEs-modified protein (23Kirstein M. Aston C. Hintz R. Vlassara H. J. Clin. Invest. 1992; 90: 439-446Crossref PubMed Scopus (237) Google Scholar). Saishoji et al. (24Saishoji T. Higashi T. Ikeda K. Sano H. Jinnouchi Y. Ogawa M. Horiuchi S. Biochem. Biophys. Res. Commun. 1995; 217: 278-285Crossref PubMed Scopus (13) Google Scholar) also suggested that AGEs-modified bovine serum albumin (BSA) stimulated the activity of urokinase-type plasminogen activator through the scavenger receptor on RAW 246.7 cell line. Schmidt et al. (25Schmidt A.M. Hori O. Chen J.X. Li J.F. Crandall J. Zhang J.H. Cao R. Yan S.D. Brett J. Stern D. J. Clin. Invest. 1993; 96: 1395-1403Crossref Scopus (813) Google Scholar) recently reported that the recognition of AGEs by receptors for AGEs, so-called RAGE, leads to the expression of VCAM-1. The interaction between AGEs and cells in the blood or vascular tissue as mentioned above may consequently cause angiopathy. In fact, several lines of evidence have shown the existence of AGEs in vascular lesions (11Nakamura Y. Horii Y. Nishino T. Shiiki H. Sakaguchi Y. Kagoshima T. Dohi K. Makita Z. Vlassara H. Bucala R. Am. J. Pathol. 1993; 143: 1649-1656PubMed Google Scholar, 12Kume S. Takeya M. Mori T. Araki N. Suzuki H. Horiuchi S. Kodama T. Miyauchi Y. Takahashi K. Am. J. Pathol. 1995; 147: 654-667PubMed Google Scholar). However, the exact moiety of AGEs-modified protein recognized by the cells is not yet known.In the present study, we investigated a pyrraline-modified protein that had been demonstrated to localize at thickened intima of arteriosclerotic lesion in the kidney of diabetic patients (15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). We examined the effect of modification of albumin by pyrraline on the degradation of the protein by macrophage-like cell line, P388D1 cells. This cell line was originally isolated by Dawe and Potter (26Dawe C. Potter M. Am. J. Pathol. 1957; 33: 603Google Scholar) from a methylcholanthrene-induced lymphoid neoplasm of a DBA/2 mouse. Subsequent investigation by Koren et al. (27Koren H.S. Handwerger B.S. Wunderlich J.R. J. Immunol. 1975; 114: 894-897PubMed Google Scholar) revealed that these cells have macrophage-like characteristics. Among these, the P388D1 cells have phagocytic activity and are rich in lysosomal vesicles.DISCUSSIONThe present study was based on the recent demonstration of pyrraline in arteriosclerotic lesions and designed to examine the relationship defining the interaction between pyrraline-modified protein and blood cells, such as macrophages, with the progress of angiopathy. The deposition of pyrraline in arteriosclerotic lesions is somewhat similar to cholesterol deposition in atherosclerotic lesions. The latter is known to be associated with the uptake of chemically modified low density lipoprotein by scavenger receptor of monocytes/macrophages (37Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). Several receptors, including scavenger receptor and RAGE, are thought to recognize AGEs-modified proteins (19Vlassara H. Brownlee M. Cerami A. Proc. Natl. Sci. U. S. A. 1985; 82: 5588-5592Crossref PubMed Scopus (287) Google Scholar, 20Schmidt A.M. Vianna M. Gerlach M. Brett J. Ryan J. Kao J. Esposito C. Hegarty H. Hurley W. Clauss M. Wang F. Pan Y-C.E. Tsang T.C. Stern D. J. Biol. Chem. 1992; 267: 14987-14997Abstract Full Text PDF PubMed Google Scholar, 21Araki N. Higashi T. Mori T. Shibayama R. Kawabe Y. Kodama T. Takahashi K. Shichiri M. Horiuchi S. Eur. J. Biochem. 1995; 230: 408-415Crossref PubMed Scopus (223) Google Scholar). Vlassara et al. (19Vlassara H. Brownlee M. Cerami A. Proc. Natl. Sci. U. S. A. 1985; 82: 5588-5592Crossref PubMed Scopus (287) Google Scholar) reported that the uptake and degradation of AGE-modified BSA is achieved thorough a high-affinity receptor on mouse macrophages. The removal of AGE-modified protein by such mechanism is an attractive hypothesis of tissue remodeling. However, accumulation of AGEs-modified BSA was still present in the cells examined by Vlassara et al. (19Vlassara H. Brownlee M. Cerami A. Proc. Natl. Sci. U. S. A. 1985; 82: 5588-5592Crossref PubMed Scopus (287) Google Scholar), presumably due to an inefficient degradation compared with albumin uptake. Furthermore, these investigators also reported accumulation of AGEs-modified nerve myelin in macrophages and proposed that the interaction between AGEs-modified nerve protein and macrophages may initiate demyelination in diabetes (38Vlassara H. Brownlee M. Cerami A. J. Exp. Med. 1984; 160: 197-207Crossref PubMed Scopus (116) Google Scholar). In this regard, the exact structure of AGEs moiety recognized by the receptor is still unknown. Furthermore, the exact effect of AGEs modification is complicated by the presence of heterogeneous AGEs. Therefore, we focused in the present study on pyrraline-modified protein as a ligand, since its structure had been identified. Using pyrraline as a model of AGEs, estimation of the involvement of the modification became more accessible. The P388D1 used in this study is an established macrophage-like cell line with phagocytic activity. Westwood et al. (39Westwood M.E. McLellan A.C. Thornalley P.J. J. Biol. Chem. 1994; 269: 32293-32298Abstract Full Text PDF PubMed Google Scholar) recently showed the existence of receptor recognizing methylglyoxal-modified protein on P388D1 cell surface. Although we attempted to identify a high-affinity cell surface receptor against pyrraline, we were unable to find such receptor in the range of ligand concentration examined (data not shown). Since P388D1 cells are able to phagocyte albumin themselves, pyrraline-modified BSA may not be necessarily recognized at the pyrraline moiety. However, the possible existence of a lower-affinity receptor cannot be excluded completely at present.Our results showed that pyrraline-modified albumin was resistant to degradation by the cells. This finding suggests that pyrraline-modified albumin may have affected lysosomal proteolytic function of P388D1 cells. Alternatively, the susceptibility of albumin to proteolysis was modified by pyrraline. examine the we compared lysosomal function of cells with and pyrraline-modified Our results showed that the of lysosomal and were not the other the susceptibility of pyrraline-modified albumin to enzymatic proteolysis by was reduced in is known to be by and in the Scopus Google Scholar). Since is to examine the susceptibility of pyrraline-modified albumin to lysosomal proteolytic we by studies showed that pyrraline-modified albumin was resistant to by as as are with the known that its enzymatic as is modified by AGEs J. Clin. Invest. PubMed Scopus Google Scholar, M. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). Furthermore, the of pyrraline modification observed at approximately BSA was to the against enzymatic Although the reduced was on the of the modification by an BSA showed more effect an the of of of pyrraline-modified BSA by or in was to that observed in the degradation of pyrraline-modified albumin in using P388D1 cells. we that the cause of of degradation by phagocytes is more to be a in the susceptibility to lysosomal proteolytic in P388D1 cells. It is that the of degradation leads to accumulation of pyrraline-modified albumin in the cells. In addition, the recent finding of pyrraline accumulation in lesions of such as and suggests a role for AGEs, including pyrraline, in the of the lesions S. Miyata S. Yan Stern D. Sayre L.M. Monnier V.M. Proc. Natl. Sci. U. S. A. 1994; PubMed Scopus Google present results also showed accumulation of pyrraline-modified albumin in human monocytes as as P388D1 cell line. Plasma pyrraline levels were shown to be elevated in diabetic rats and humans as determined by ELISA using antibody to pyrraline (15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). the pyrraline modification per of albumin from the diabetic showed of blood glucose more modification such as Since glycation not to albumin the of the modification in may be more the In fact, AGEs formed in albumin in vivo is to be very In to pyrraline, pentosidine has been reported to in diabetic plasma J. D.R. Monnier V.M. Diabetes. 1992; PubMed Google Scholar, S. Monnier V.M. Clin. Chem. 1994; PubMed Scopus Google Scholar). other AGEs, Makita et al. Z. Vlassara H. Cerami A. Bucala R. J. Biol. Chem. 1992; 267: 5133-5138Abstract Full Text PDF PubMed Google Scholar) reported that the levels of AGE-modified serum protein from diabetic patients with were elevated when determined by using antibody not with pyrraline or Although structures of the AGEs recognized by the antibody still to be that serum protein is modified by AGEs as as pyrraline in diabetic Since the in the susceptibility of pyrraline-modified albumin to lysosomal observed in the present study, to the modification of the may be that other pyrraline also have similar on enzymatic degradation of Kato et al. H. F. Biol. Chem. 1987; Scopus Google Scholar) showed that of human serum albumin with glucose in the in lysine by to pyrraline-modified and N. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) also that lysine were the glycation by amino of albumin from diabetic et al. A. D. R. S. R. 1995; PubMed Scopus Google Scholar) more determined the of glycation in serum albumin from diabetic using a They that the of modified in a of albumin was from to that the of modification of albumin in the present study is in the AGE-modified proteins in the of diabetic patients may in monocytes/macrophages in vivo and J.L. Diabetes. 1984; 33: PubMed Scopus Google Scholar) also demonstrated an effect of glycation on the degradation of in They also suggested that the effect was due to a modification in lysine of to protein the modification of lysine by AGEs may be more since the is In fact, the levels of in of diabetic patients are also as reported by Bucala et al. R. Makita Z. T. Cerami A. Vlassara H. Proc. Natl. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). The exact of the of these cells following accumulation of AGE-modified protein is the that modification of is a for modification of albumin by AGEs, such as pyrraline, may be also involved in vascular The present may to the involved in modification of proteins in vivo causing a in and of diabetic INTRODUCTIONMaillard reaction (glycation) is thought to play a role in the pathogenesis of angiopathy in diabetes and aging process (1Cohen M.P. Diabetes and Protein Glycosylation. Springer-Verlag, New York1986Crossref Google Scholar, 2Brownlee M. Cerami A. Vlassara H. N. Engl. J. Med. 1988; 318: 1315-1321Crossref PubMed Scopus (2324) Google Scholar, 3Monnier V.M. Baynes J.W. The Maillard Reaction in Aging Diabetes and Nutrition. Alan R. Liss, New York1989Google Scholar). The advanced stage of this reaction that leads to the formation of advanced glycation end products (AGEs) 1The abbreviations used are: AGEadvanced glycation end productBSAbovine serum albuminPyr-BSApyrraline-modified BSARAGEreceptor for AGEVCAM-1vascular cell adhesion molecule-1PBSphosphate-buffered salineELISAenzyme-linked immunosorbent assay. is very complex due to several possible metabolic pathways. Despite this complexity, the structures of several AGEs have been recently described, such as that of pyrraline (4Njoroge F.G. Sayre L.M. Monnier V.M. Carbohydr. Res. 1987; 167: 211-220Crossref PubMed Scopus (115) Google Scholar), pentosidine (5Sell D.R. Monnier V.M. J. Biol. Chem. 1989; 264: 21597-21602Abstract Full Text PDF PubMed Google Scholar), cross-line (6Nakamura K. Hasegawa T. Fukunaga Y. Ienaga K. Chem. Soc. Chem. Commun. 1992; 14: 992-994Crossref Google Scholar), and pyrropyridine (7Hayase F. Hinuma H. Asano M. Kato H. Arai S. Biosci. Biotechnol. Biochem. 1994; 58: 1936-1937Crossref Google Scholar). Carboxymethyllysine is also formed by oxidation of Amadori products (8Dunn J.A. McCance D.R. Thorpe S.R. Lyons T.J. Baynes J.W. Biochemistry. 1991; 30: 1205-1210Crossref PubMed Scopus (284) Google Scholar). Pyrraline is one of the AGEs derived from the reaction of glucose with the lysine amino group on proteins. Several investigators have demonstrated the preferential accumulation of AGEs in diabetic tissues (9Nakayama H. Mitsuhashi T. Kuwajima S. Aoki S. Kuroda Y. Itoh T. Nakagawa S. Diabetes. 1993; 42: 345-350Crossref PubMed Scopus (134) Google Scholar, 10Mitsuhashi T. Nakayama H. Itoh T. Kuwajima S. Aoki S. Atsumi T. Koike T. Diabetes. 1993; 42: 826-832Crossref PubMed Scopus (95) Google Scholar, 11Nakamura Y. Horii Y. Nishino T. Shiiki H. Sakaguchi Y. Kagoshima T. Dohi K. Makita Z. Vlassara H. Bucala R. Am. J. Pathol. 1993; 143: 1649-1656PubMed Google Scholar, 12Kume S. Takeya M. Mori T. Araki N. Suzuki H. Horiuchi S. Kodama T. Miyauchi Y. Takahashi K. Am. J. Pathol. 1995; 147: 654-667PubMed Google Scholar, 13Makita Z. Vlassara H. Cerami A. Bucala R. J. Biol. Chem. 1992; 267: 5133-5138Abstract Full Text PDF PubMed Google Scholar). Plasma pyrraline levels are also elevated in diabetic rats and humans as determined by ELISA using antibody to pyrraline (14Hayase F. Nagaraj R.H. Miyata S. Njoroge F.G. Monnier V.M. J. Biol. Chem. 1989; 264: 3758-3764Abstract Full Text PDF PubMed Google Scholar, 15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). In addition, Porterootin et al. (16Porterootin M. Nagaraj R.H. Monnier V.M. Biochim. Biophys. Acta. 1995; 1247: 74-80Crossref PubMed Scopus (87) Google Scholar) demonstrated the existence of pyrraline in vivo using high performance liquid chromatography. They also showed that pyrraline in vivo could react with other amino acids on proteins to form cross-links (17Nagaraj R.H. Porterootin M. Monnier V.M. Arch. Biochem. Biophys. 1996; 325: 152-158Crossref PubMed Scopus (52) Google Scholar). It was also demonstrated that pyrraline is found in vascular lesions of diabetic and elderly subjects using immunohistochemical techniques (15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). Among several pathways forming pyrraline, highly reactive dicarbonyl compounds, such as 3-deoxyglucosone, were identified as precursors reacting with free amino groups to form pyrraline (14Hayase F. Nagaraj R.H. Miyata S. Njoroge F.G. Monnier V.M. J. Biol. Chem. 1989; 264: 3758-3764Abstract Full Text PDF PubMed Google Scholar, 15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). We recently reported that plasma 3-deoxyglucosone levels are elevated in diabetic rats using specific high performance liquid chromatography assay (18Yamada H. Miyata S. Igaki N. Yatabe H. Miyauchi Y. Ohara T. Sakai M. Shoda H. Oimomi M. Kasuga M. J. Biol. Chem. 1994; 269: 20275-20280Abstract Full Text PDF PubMed Google Scholar).AGEs are also known to alter the structural and functional properties of proteins. Furthermore, a pathological role of the interaction between AGEs-modified proteins and cells for diabetic complications has been recently proposed. Several cell surface proteins are thought to recognize AGEs (19Vlassara H. Brownlee M. Cerami A. Proc. Natl. Sci. U. S. A. 1985; 82: 5588-5592Crossref PubMed Scopus (287) Google Scholar, 20Schmidt A.M. Vianna M. Gerlach M. Brett J. Ryan J. Kao J. Esposito C. Hegarty H. Hurley W. Clauss M. Wang F. Pan Y-C.E. Tsang T.C. Stern D. J. Biol. Chem. 1992; 267: 14987-14997Abstract Full Text PDF PubMed Google Scholar, 21Araki N. Higashi T. Mori T. Shibayama R. Kawabe Y. Kodama T. Takahashi K. Shichiri M. Horiuchi S. Eur. J. Biochem. 1995; 230: 408-415Crossref PubMed Scopus (223) Google Scholar). Furthermore, interaction between AGEs and these proteins induces a variety of secondary effects. Vlassara et al. (22Vlassara H. Brownlee M. Manogue K.R. Dinarello C. Pasagian A. Science. 1988; 240: 1546-1548Crossref PubMed Scopus (530) Google Scholar) demonstrated that macrophages secrete cytokines, such as tumor necrosis factor and interleukin-1, following the recognition of AGEs through specific receptors. They also showed the induction of insulin-like growth factor in human monocytes by AGEs-modified protein (23Kirstein M. Aston C. Hintz R. Vlassara H. J. Clin. Invest. 1992; 90: 439-446Crossref PubMed Scopus (237) Google Scholar). Saishoji et al. (24Saishoji T. Higashi T. Ikeda K. Sano H. Jinnouchi Y. Ogawa M. Horiuchi S. Biochem. Biophys. Res. Commun. 1995; 217: 278-285Crossref PubMed Scopus (13) Google Scholar) also suggested that AGEs-modified bovine serum albumin (BSA) stimulated the activity of urokinase-type plasminogen activator through the scavenger receptor on RAW 246.7 cell line. Schmidt et al. (25Schmidt A.M. Hori O. Chen J.X. Li J.F. Crandall J. Zhang J.H. Cao R. Yan S.D. Brett J. Stern D. J. Clin. Invest. 1993; 96: 1395-1403Crossref Scopus (813) Google Scholar) recently reported that the recognition of AGEs by receptors for AGEs, so-called RAGE, leads to the expression of VCAM-1. The interaction between AGEs and cells in the blood or vascular tissue as mentioned above may consequently cause angiopathy. In fact, several lines of evidence have shown the existence of AGEs in vascular lesions (11Nakamura Y. Horii Y. Nishino T. Shiiki H. Sakaguchi Y. Kagoshima T. Dohi K. Makita Z. Vlassara H. Bucala R. Am. J. Pathol. 1993; 143: 1649-1656PubMed Google Scholar, 12Kume S. Takeya M. Mori T. Araki N. Suzuki H. Horiuchi S. Kodama T. Miyauchi Y. Takahashi K. Am. J. Pathol. 1995; 147: 654-667PubMed Google Scholar). However, the exact moiety of AGEs-modified protein recognized by the cells is not yet known.In the present study, we investigated a pyrraline-modified protein that had been demonstrated to localize at thickened intima of arteriosclerotic lesion in the kidney of diabetic patients (15Miyata S. Monnier V.M. J. Clin. Invest. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). We examined the effect of modification of albumin by pyrraline on the degradation of the protein by macrophage-like cell line, P388D1 cells. This cell line was originally isolated by Dawe and Potter (26Dawe C. Potter M. Am. J. Pathol. 1957; 33: 603Google Scholar) from a methylcholanthrene-induced lymphoid neoplasm of a DBA/2 mouse. Subsequent investigation by Koren et al. (27Koren H.S. Handwerger B.S. Wunderlich J.R. J. Immunol. 1975; 114: 894-897PubMed Google Scholar) revealed that these cells have macrophage-like characteristics. Among these, the P388D1 cells have phagocytic activity and are rich in lysosomal