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• W2000607281 abstract "Lysosomal membranes contain two highly glycosylated proteins, designated LAMP-1 and LAMP-2, as major components. LAMP-1 and LAMP-2 are structurally related. To investigate the physiological role of LAMP-1, we have generated mice deficient for this protein. LAMP-1-deficient mice are viable and fertile. In LAMP-1-deficient brain, a mild regional astrogliosis and altered immunoreactivity against cathepsin-D was observed. Histological and ultrastructural analyses of all other tissues did not reveal abnormalities. Lysosomal properties, such as enzyme activities, lysosomal pH, osmotic stability, density, shape, and subcellular distribution were not changed in comparison with controls. Western blot analyses of LAMP-1-deficient and heterozygote tissues revealed an up-regulation of the LAMP-2 protein pointing to a compensatory effect of LAMP-2 in response to the LAMP-1 deficiency. The increase of LAMP-2 was neither correlated with an increase in the level oflamp-2 mRNAs nor with increased half-life time of LAMP-2. This findings suggest a translational regulation of LAMP-2 expression. Lysosomal membranes contain two highly glycosylated proteins, designated LAMP-1 and LAMP-2, as major components. LAMP-1 and LAMP-2 are structurally related. To investigate the physiological role of LAMP-1, we have generated mice deficient for this protein. LAMP-1-deficient mice are viable and fertile. In LAMP-1-deficient brain, a mild regional astrogliosis and altered immunoreactivity against cathepsin-D was observed. Histological and ultrastructural analyses of all other tissues did not reveal abnormalities. Lysosomal properties, such as enzyme activities, lysosomal pH, osmotic stability, density, shape, and subcellular distribution were not changed in comparison with controls. Western blot analyses of LAMP-1-deficient and heterozygote tissues revealed an up-regulation of the LAMP-2 protein pointing to a compensatory effect of LAMP-2 in response to the LAMP-1 deficiency. The increase of LAMP-2 was neither correlated with an increase in the level oflamp-2 mRNAs nor with increased half-life time of LAMP-2. This findings suggest a translational regulation of LAMP-2 expression. Lysosomes are membrane-bound organelles with an acidic internal milieu containing hydrolytic enzymes for degradation of proteins, lipids, nucleic acids, and saccharides. The membrane limiting the lysosomal compartment has multiple functions. It is responsible for acidification of the interior, sequestration of the active lysosomal enzymes (1Mellmann I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar), transport of degradation products from the lysosomal lumen to the cytoplasm, and regulation of fusion and fission events between lysosomes themselves and other organelles (2Peters C. von Figura K. FEBS Lett. 1994; 346: 108-114Crossref PubMed Scopus (83) Google Scholar, 3Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Abstract Full Text PDF PubMed Google Scholar). The lysosomal membranes contain several highlyN-glycosylated proteins among which the best known are LAMP-1 and LAMP-2. These two glycoproteins are structurally similar and evolutionary related (4Granger B.L. Green S.A. Gabel C.A. Howe C.L. Mellman I. Helenius A. J. Biol. Chem. 1990; 265: 12036-12043Abstract Full Text PDF PubMed Google Scholar). Alignment data suggest that chicken lysosome-endosome-plasma membrane 100 (5Fambrough D.M. Takeyasu K. Lippincott-Schwartz J. Siegel N.R. J. Cell Biol. 1988; 106: 61-67Crossref PubMed Scopus (73) Google Scholar), rat lysosomal membrane glycoprotein 120 (6Howe C.L. Granger B.L. Hull M. Green S.A. Gabel C.A. Helenius A. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7577-7581Crossref PubMed Scopus (105) Google Scholar) (also described as rat lysosomal membrane glycoprotein 107 (7Himeno M. Nogushi Y. Sasaki H. Tanaka Y. Furuno K. Kono A. Sasaki Y. Kato K. FEBS Lett. 1989; 244: 351-356Crossref PubMed Scopus (41) Google Scholar)), mouse lysosomal-associated membrane protein-1 (8Chen J.W. Cha Y. Yuksel K.U. Gracy R.W. August J.T. J. Biol. Chem. 1988; 263: 8754-8758Abstract Full Text PDF PubMed Google Scholar), and human lysosomal-associated membrane protein A (9Viitala J. Carlsson S.R. Siebert P.D. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3743-3747Crossref PubMed Scopus (73) Google Scholar) also described as human lysosomal-associated membrane protein-1 (10Fukuda M. Viitala J. Matteson J. Carlsson S.R. J. Biol. Chem. 1988; 263: 18920-18928Abstract Full Text PDF PubMed Google Scholar) are species specific versions of the same protein designated as LAMP-1 or lysosomal membrane glycoprotein A (4Granger B.L. Green S.A. Gabel C.A. Howe C.L. Mellman I. Helenius A. J. Biol. Chem. 1990; 265: 12036-12043Abstract Full Text PDF PubMed Google Scholar, 11Kornfeld S. Mellman I. Ann. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1239) Google Scholar). LAMP-1 is composed of a large luminal portion, which is separated by a proline-rich hinge region in two disulfide-containing domains, a single transmembrane-spanning segment and a short cytoplasmic tail of 11 amino acids (9Viitala J. Carlsson S.R. Siebert P.D. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3743-3747Crossref PubMed Scopus (73) Google Scholar). The latter contains a Gly-Tyr motif critical for transport to lysosomes (12Guarnieri F.G. Arterburn L.M. Penno M.B. Cha Y. August J.T. J. Biol. Chem. 1993; 268: 1941-1946Abstract Full Text PDF PubMed Google Scholar, 13Höning S. Hunziker W. J. Cell Biol. 1995; 128: 464-473Crossref Scopus (117) Google Scholar). The polypeptide of LAMP-1 contains 382 amino acids, corresponding to about 42 kDa. The apparent size of the newly synthesized LAMP-1 is 92,000. The increase in size is due to N- andO-glycosylation. The luminal portion of the polypeptide chain contains 16–20 potential N-glycosylation sites, most or all of which are utilized. Processing of the oligosaccharides converts the LAMP-1 precursor in a family of mature forms differing in size from 110 to 140 kDa. Part of the glycans are of the polylactosamine type, and LAMP-1 is one of the major carriers for poly-N-acetyllactosamines in cells. Interestingly, the content of poly-N-acetyllactosamines in LAMP-1 correlates with differentiation (14Lee N. Wang W.C. Fukuda M. J. Biol. Chem. 1990; 265: 20476-20487Abstract Full Text PDF PubMed Google Scholar, 15Carlsson S.R. Fukuda M. J. Biol. Chem. 1989; 264: 20526-20531Abstract Full Text PDF PubMed Google Scholar) and metastatic potential (16Youakim A. Romero P.A. Yee K. Carlsson S.R. Fukuda M. Herscovics A. Cancer Res. 1989; 49: 6889-6895PubMed Google Scholar, 17Yamashita K. Ohkura T. Tachibana Y. Takasaki S. Kobata A. J. Biol. Chem. 1984; 259: 10834-10840Abstract Full Text PDF PubMed Google Scholar, 18Pierce M. Arango J. J. Biol. Chem. 1986; 261: 10772-10777Abstract Full Text PDF PubMed Google Scholar, 19Yousefi S. Higgins E. Daoling Z. Pollex-Krüger A. Hindsgaul O. Dennis J.W. J. Biol. Chem. 1991; 266: 1772-1782Abstract Full Text PDF PubMed Google Scholar) of tumor cells. The lamp-1 gene is ubiquitously expressed (20Zot A.S. Fambrough D.M. J. Biol. Chem. 1990; 265: 20988-20995Abstract Full Text PDF PubMed Google Scholar) with somewhat higher levels in spleen, liver, and kidney (20Zot A.S. Fambrough D.M. J. Biol. Chem. 1990; 265: 20988-20995Abstract Full Text PDF PubMed Google Scholar, 21Laferte S. Dennis J.W. Biochem. J. 1989; 259: 569-576Crossref PubMed Scopus (52) Google Scholar, 22Heffernan M. Yousefi S. Dennis J.W. Cancer Res. 1989; 49: 6077-6084PubMed Google Scholar). In P388 macrophages, LAMP-1 comprises about 0.1% of total cell protein, corresponding to about 2 × 106 LAMP-1 molecules/cell (23Chen J.W. Pan W. D'Souza M.P. August J.T. Arch. Biochem. Biophys. 1985; 239: 574-586Crossref PubMed Scopus (90) Google Scholar). The collective abundance of both LAMP-1 and LAMP-2 has been estimated to be high enough to form a nearly continuous carbohydrate coat on the inner surface of the lysosomal membrane (3Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Abstract Full Text PDF PubMed Google Scholar, 4Granger B.L. Green S.A. Gabel C.A. Howe C.L. Mellman I. Helenius A. J. Biol. Chem. 1990; 265: 12036-12043Abstract Full Text PDF PubMed Google Scholar). Although LAMP-1 is distributed within the cell primarily in the lysosome, under certain circumstances, e.g. after platelet activation (24Febbraio M. Silverstein R.L. J. Biol. Chem. 1990; 265: 18531-18537Abstract Full Text PDF PubMed Google Scholar), during granulocytic differentiation and activation (14Lee N. Wang W.C. Fukuda M. J. Biol. Chem. 1990; 265: 20476-20487Abstract Full Text PDF PubMed Google Scholar, 25Dahlgren C. Carlsson S.R. Karlsson A. Lundquist H. Sjölin C. Biochem. J. 1995; 311: 667-674Crossref PubMed Scopus (72) Google Scholar), and on cytotoxic T lymphocytes (26Peters P.J. Borst J. Oorschot V. Fukuda M. Krähenbühl O. Tschopp J. Slot J.W. Geuze H.J. J. Exp. Med. 1991; 173: 1099-1109Crossref PubMed Scopus (528) Google Scholar) it is also found at the cell surface. LAMP-1 was also found on the cell surface of highly metastatic tumor cells (3Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Abstract Full Text PDF PubMed Google Scholar, 16Youakim A. Romero P.A. Yee K. Carlsson S.R. Fukuda M. Herscovics A. Cancer Res. 1989; 49: 6889-6895PubMed Google Scholar, 17Yamashita K. Ohkura T. Tachibana Y. Takasaki S. Kobata A. J. Biol. Chem. 1984; 259: 10834-10840Abstract Full Text PDF PubMed Google Scholar, 18Pierce M. Arango J. J. Biol. Chem. 1986; 261: 10772-10777Abstract Full Text PDF PubMed Google Scholar, 19Yousefi S. Higgins E. Daoling Z. Pollex-Krüger A. Hindsgaul O. Dennis J.W. J. Biol. Chem. 1991; 266: 1772-1782Abstract Full Text PDF PubMed Google Scholar). It was suggested that cell surface-expressed LAMP can serve as ligand for selectins (21Laferte S. Dennis J.W. Biochem. J. 1989; 259: 569-576Crossref PubMed Scopus (52) Google Scholar, 27Sawada R. Jardine K.A. Fukuda M. J. Biol. Chem. 1993; 268: 9014-9022Abstract Full Text PDF PubMed Google Scholar) and mediate cell-cell adhesion/recognition events (28Acevedo-Schermerhorn C. Gray-Bablin J. Gama R. McCormick P.J. Exp. Cell Res. 1997; 236: 510-518Crossref PubMed Scopus (6) Google Scholar). At present there are hardly any data as to the in vivofunctions of lysosomal membrane glycoproteins. To get an approach for the analysis of the physiological role of this group of proteins, we have inactivated the lamp-1 gene in the mouse by targeted disruption. Although LAMP-1-deficient mice lack an overt phenotype, an increased expression of the related LAMP-2 was observed in LAMP-1 homozygote and heterozygote deficient tissues. These findings strongly suggest that LAMP-2 can at least partially compensate for the loss of LAMP-1 and that the expression of both proteins is tightly regulatedin vivo. An EMBL3–129SV mouse phage library from Stratagene Inc. (La Jolla, CA) was screened with a 550-bp 1The abbreviations used are: bp, base pair(s); kbp, kilobase pair(s); kb, kilobase(s); PCR, polymerase chain reaction; PBS, phosphate-buffered saline; BSA, bovine serum albumingenomic amplification product of mouse lamp-1 corresponding to cDNA positions 118–350 (8Chen J.W. Cha Y. Yuksel K.U. Gracy R.W. August J.T. J. Biol. Chem. 1988; 263: 8754-8758Abstract Full Text PDF PubMed Google Scholar). The probe contained exon 2 and exon 3 of mouselamp-1 interrupted by a small intron. The isolated mouselamp-1 phage clone mouse LAMP-1/1 contained the 5′-region of the gene with exons 2–5. DNA sequence analyses revealed complete sequence identity of four exons with the nucleotide sequence of the mouse lamp-1 cDNA (data not shown). For construction of a targeting vector, a 5.3-kbp KpnI DNA restriction fragment of lamp-1 covering exons 2 and 3 (see Fig. 1 A,II) was subcloned into the plasmid vector pBluescript SKII+ (Stratagene). The neo expression cassette from pMC1neopA (Ref. 29Thomas K.R. Cappechi M.R. Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1835) Google Scholar; Stratagene) was inserted as a BamHI DNA restriction fragment into a BglII restriction site located in exon 3 of the KpnI fragment (nucleotide position 323 of the lamp-1 cDNA; amino acid 107 of 382 amino acids; Ref.8Chen J.W. Cha Y. Yuksel K.U. Gracy R.W. August J.T. J. Biol. Chem. 1988; 263: 8754-8758Abstract Full Text PDF PubMed Google Scholar). The insertion of the neo cassette introduces a premature translational stop codon into the open reading frame of thelamp-1 gene. Additionally, for negative selection with gancyclovir, a thymidine kinase cassette was inserted at the 3′ site of the KpnI fragment. The targeting vector was linearized withXhoI and introduced into the ES cell line E14–1 by electroporation. ES cells were cultured as described by Kösteret al. (30Köster A. Saftig P. Matzner U. von Figura K. Peters C. Pohlmann R. EMBO J. 1993; 12: 5219-5223Crossref PubMed Scopus (77) Google Scholar). G418- and gancyclovir-resistant colonies were screened by Southern blot analysis of DNA digested withHindIII and hybridized with the 3′ probe (Fig.1 A). Two ES cell clones with homologous recombination were confirmed by digesting DNA with BglII and hybridization with the 5′ probe (Fig. 1 A). The mutated ES lines were microinjected into blastocysts of C57BL/6J mice. Chimeric males were mated to C57BL/6J females. Mice were genotyped for thelamp-1 gene mutation by Southern blot analysis ofHindIII-digested genomic DNA, using the 3′ probe or by PCR analyses using a neomycin-specific PCR (31Saftig P. Peters C. von Figura K. Craessaerts K. van Leuven F. de Strooper B. J. Biol. Chem. 1996; 271: 27241-27244Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and an exon-specific PCR with primers (LAMP-1/4B 5′-cttgatgtcagttgtggaatccat-3′ and LAMP-1/3E 5′-tttccctgccagcctctgcagaag-3′) flanking the exon used for interruption. Homozygous mutant mice were obtained by mating heterozygous or homozygous mutant mice. For initial phenotype testing, littermates of the F2 and F3 generations were used. In later experiments, offspring of homozygote-deficient mice were compared with age- and sex-matched offspring of control mice. The mice were kept in a conventional animal facility at the Zentrum für Biochemie und Molekulare Zellbiologie, Universität Göttingen (Göttingen, Germany). Total RNA of liver and kidney from 3-month-old mice was prepared using the Qiagen RNeasy system. Ten micrograms of total RNA were separated in a formaldehyde agarose gel and processed as described by Isbrandt et al. (32Isbrandt D. Arlt G. Brooks D.A. Hopwood J.J. von Figura K. Peters C. Am. J. Hum. Genet. 1994; 54: 454-463PubMed Google Scholar). Filters were hybridized with a lamp-1 5′ probe used for genomic library screening, a lamp-2 cDNA probe (33Cha Y. Holland S.M. August J.T. J. Biol. Chem. 1990; 265: 5008-5013Abstract Full Text PDF PubMed Google Scholar), and a 280-bp cDNA fragment from glyceraldehyde-3-phosphate dehydrogenase (34Lyons K. Graycar J.L. Lee A. Hashmi S. Lindquist P.B. Chen E.Y. Hogan B.L.M. et al.Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4554-4558Crossref PubMed Scopus (151) Google Scholar). Hybridization and washing of filters were performed as described by Lehmann et al. (35Lehmann L.E. Eberle W. Krull S. Prill V. Schmidt B. Sander C. von Figura K. Peters C. EMBO J. 1992; 11: 4391-4399Crossref PubMed Scopus (53) Google Scholar). Expression of LAMP-1, LAMP-2, and LIMP-2 (lysosomal membrane glycoprotein 85) was analyzed in tissue homogenates (liver, kidney, spleen, brain, heart, adult, and embryonic fibroblasts). Frozen tissues were homogenized in Tris-buffered saline (w/v; 1:9) at 4 °C using an Ultra-Turrax, analyzed for protein (36Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), adjusted to 1% Triton X-100, and used for enzyme determination (see below) and Western blot analysis. For LAMP-1, LAMP-2, and LIMP-2, 100 μg of protein of tissue homogenate was subjected to SDS-PAGE (7.5% polyacrylamide) under reducing conditions. Proteins were transferred to a polyvinylidene difluoride membrane (Schleicher und Schüll, Dassel, Germany), which was subsequently blocked with 10 mmPBS, pH 7.4, 0.05% Triton X-100, 5% milk powder (blocking buffer) for 1 h at 37 °C. The blot was incubated overnight at 4 °C with a monoclonal anti-mouse LAMP-1 antibody (1D4B, Developmental Studies Hybridoma Bank, Iowa City, IA) in a 1:250 dilution, an anti-mouse LAMP-2 antibody (Abl 93; Developmental Studies Hybridoma Bank) in a 1:100 dilution, and a polyclonal anti-rat LIMP-2 (37Okazaki I. Himeno M. Ezaki J. Ishikawa T. Kato K. J. Biochem. (Tokyo). 1992; 111: 763-769Crossref PubMed Scopus (36) Google Scholar) antibody in a 1:400 dilution, respectively. Membranes were washed six times for 5 min in 10 mm PBS, pH 7.4, 0.1% Tween 20. Subsequently, incubation with horseradish peroxidase-coupled anti-rat antibody (1:7500 for LAMP-1 and LAMP-2) or with horseradish peroxidase-coupled anti-rabbit antibody (1:20,000 for LIMP-2) was performed for 1 h at room temperature followed by washing six times for 5 min in 10 mm PBS, pH 7.4, 0.1% Tween 20. Blots were finally analyzed using the ECL Detection System (Amersham Pharmacia Biotech). Quantification was performed by densitometry (Hewlett-Packard Scan Jet 4c/T; WinCam 2.2). Mouse embryonic fibroblasts, mouse adult fibroblasts, and peritoneal macrophages were grown on glass coverslips for 1 day. The cells were fixed with methanol or paraformaldehyde with 0.5% saponin. Cathepsin-D was immunostained using a rabbit antiserum (38Pohlmann R. Boecker M.W.C. von Figura K. J. Biol. Chem. 1995; 270: 27311-27318Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), LAMP-1 and LAMP-2 were immunostained using a monoclonal anti-mouse rat hybridoma medium (1D4B and Abl 93; Developmental Studies Hybridoma Bank), and LIMP-2 was immunostained using a polyclonal rabbit anti rat-lysosomal membrane glycoprotein 85 antiserum (37Okazaki I. Himeno M. Ezaki J. Ishikawa T. Kato K. J. Biochem. (Tokyo). 1992; 111: 763-769Crossref PubMed Scopus (36) Google Scholar). The primary antibodies were detected with goat anti-rabbit Texas Red, goat anti-rat Texas Red, and goat anti-rat 5-(4,6-[dichlootriazin-2-yl]amino)fluorescein (Dianova, GmbH, Hamburg, Germany). After embedding in Mowiol (Calbiochem-Novabiochem GmbH), fluorescence was examined using a confocal laser scanning microscope (LSM 2; ZEISS, Oberkochen, Germany) with the filter combination described by Schulze-Garg et al. (39Schulze-Garg C. Boker C. Nadimpalli S.K. von Figura K. Hille-Rehfeld A. J. Cell Biol. 1993; 122: 541-551Crossref PubMed Scopus (19) Google Scholar). Tissue homogenates of liver, kidney, and fibroblasts were prepared in 0.25m sucrose buffered with 3 mm imidazole/HCl, pH 7.4. 0.8 ml of a postnuclear supernatant was applied onto 11.2 ml of a 20% Percoll solution (40Lemanski P. Gieselmann V. Hasilik A. von Figura K. J. Biol. Chem. 1984; 259: 10129-10135PubMed Google Scholar) and centrifuged for 30 min at 20,000 rpm in the vertical rotor VTi 65.1 (Beckman). Density and β-hexosaminidase activity were determined in each of 12 fractions collected. Fractions corresponding to dense vesicles were pooled according to the distribution of β-hexosaminidase activity and centrifuged for 1 h at 100,000 × g. The membraneous layer (lysosome-enriched pellet) floating above the pelleted silica was collected (41Bresciani R. von Figura K. Eur. J. Biochem. 1996; 238: 669-674Crossref PubMed Scopus (29) Google Scholar). 50 μl of the lysosome-enriched pellet were suspended in 250 μl of 0.25 m sucrose, pH 7.0, and 0.25 m glucose, pH 7.0, respectively. The reactions were incubated at 37 °C, and samples (30 μl) were withdrawn for assay at 0, 5, 10, 15, and 20 min. Immediately after the withdrawal, the samples were suspended in 270 μl of ice-cold 0.25 msucrose solution and subjected to centrifugation for 10 min at 30,000 rpm and 4 °C in a Beckman TL-100 centrifuge. 150 μl of the resulting supernatants were collected and kept at −20 °C until β-hexosaminidase enzyme activity measurements. To calculate the total β-hexosaminidase activity (100% of the possible β-hexosaminidase enzyme activity), one sample was incubated in 0.25 msucrose in the presence of 1% Triton X-100. β-hexosaminidase activities in the supernatants were calculated as percentages of total activity. Lysosomal enzymes were detected using fluorimetric assays as described by Köster et al. (42Köster A. von Figura K. Pohlmann R. Eur. J. Biochem. 1994; 224: 685-689Crossref PubMed Scopus (38) Google Scholar). Arylsulfatase A was measured usingp-nitrocatechol sulfate as substrate (43Porter M.T. Fluharty A.L. Kihara H. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 887-891Crossref PubMed Scopus (86) Google Scholar). Mouse embryonic fibroblasts and mouse adult fibroblasts were incubated in methionine-free medium for 1 h and then labeled with [35S]methionine/cysteine (Amersham Pharmacia Biotech) in the same medium containing 5% dialyzed fetal calf serum. During the following chase for 1, 2, 4, and 6 h, the medium was supplemented with 0.25 mg/ml l-methionine. Immunoprecipitation from cells and media was carried out as described previously (44Waheed A. Gottschalk S. Hille A. Krentler C. Pohlmann R. Braulke T. Hauser H. Geuze H. von Figura K. EMBO J. 1988; 7: 2351-2358Crossref PubMed Scopus (85) Google Scholar) with antisera specific for mouse cathepsin-D (38Pohlmann R. Boecker M.W.C. von Figura K. J. Biol. Chem. 1995; 270: 27311-27318Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). For LAMP-2 immunoprecipitation cells were labeled for 2 h with [35S]methionine and chased for 24, 48, 72, and 96 h, respectively. Immunoprecipitation of cells was done as described for cathepsin-D with overnight incubation of 2 μg of monoclonal antibody Abl 93 and subsequent incubation for 1 h with 20 μg of a “goat anti-rat” bridge antibody. Densitometric quantification of cathepsin-D and LAMP-2 was done with a phosphor imager (Fuji) and the program MacBas. For standard light microscopic histology and immunohistochemistry, tissues were fixed by cardiac perfusion with 4% paraformaldehyde in 0.1 m phosphate buffer and shock-frozen or fixed by perfusion with Bouin's solution diluted 1:3 with 0.1m phosphate buffer and embedded in paraffin. Sections were cut at 7 μm, dewaxed in xylene, passed through a graded series of alcohol, and washed in PBS. After blocking with 0.75% BSA and quenching of endogenous peroxidase activity with 3% H2O2 in methanol, sections were incubated with antibodies against cathepsin-D (38Pohlmann R. Boecker M.W.C. von Figura K. J. Biol. Chem. 1995; 270: 27311-27318Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar); glial fibrillary acidic protein (Dako, Hamburg, Germany); F4/80 (clone obtained from the Developmental Studies Hybridoma Bank); LAMP-1 (clone obtained from the Developmental Studies Hybridoma Bank); MHC-II (Pharmingen, Hamburg, Germany); and lectins RCA-1, GS-I B4, and Solanum tuberosum (all lectin reagents from Vector Laboratories, Inc. (Burlingame, CA). Detection of bound primary reagents was performed by either the avidin-biotin complex technique (reagents from Vector Laboratories), gold-labeled secondary antibodies followed by silver intensification (British BioCell), or tyramide signal amplification (all reagents from NEN Life Science Products, Bad Homburg, Germany). For transmission electron microscopy, animals were perfused with 6% glutaraldehyde in phosphate buffer and stored in fixative until further processing. Tissue blocks were rinsed in phosphate buffer, postfixed in OsO4 for 2 h, and embedded in Araldite or Epon 812 according to routine procedures. Ultrathin sections were collected on copper grids, contrasted with uranyl acetate and lead citrate, and observed with Zeiss EM 900 and EM 902 microscopes. Mouse fibroblasts were grown on plastic tissue culture wells until semiconfluency. The cells were fed with 5-nm gold particles coated with bovine serum albumin and diluted in serum-free Dulbecco's modified Eagle's medium to an optical density of 5.0, for 2 h. The cells were then fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4, and prepared for conventional Epon embedding. Thin sections were photographed with a Jeol 1200EX transmission electron microscope. Mouse kidneys were fixed by perfusion with 4% paraformaldehyde in 0.2 m Hepes, pH 7.4. Fixation was continued in immersion for 2 h at room temperature, and the tissue cubes were then stored in 2% paraformaldehyde. For cryosectioning, tissue cubes from the cortex were infiltrated in 18% polyvinylpyrrolidone, 1.66 m sucrose in PBS overnight at 4 °C, mounted on specimen holders, and frozen in liquid nitrogen. Semithin sections (500 nm) and thin sections (80 nm) were cut at −100 °C for immunofluorescence and immunoelectron microscopy, respectively. The sections were labeled with rat LAMP-1 (1D4B), rat LAMP-2 (Abl 93), or rabbit cathepsin-D antibodies, which were detected using goat anti-rat IgG-fluorescein, goat anti-rabbit-lissamine rhodamine (Jackson ImmunoResearch Laboratories, West Grove, PA), or rabbit anti-rat IgG (Jackson ImmunoResearch Laboratories) followed by protein A coupled to 10-nm colloidal gold (University of Utrecht, The Netherlands) or directly with protein A-gold. Semithin sections were mounted in Mowiol containing Diazobicyclooctane, and thin sections were mounted in 1.5% methyl cellulose containing 0.4% uranyl acetate. Two control and two LAMP-1-deficient mice were used for the immunocytochemical analysis. A 12-kbp genomic clone from the lamp-1 structural gene region was isolated by screening a genomic 129/SvJ mouse phage library with a genomic PCR fragment as probe containing exons 2 and 3 of lamp-1, corresponding to amino acid residues 37–114 of the LAMP-1 protein (Fig. 1 A,I; for details see “Experimental Procedures”). The targeting vector (Fig. 1 A, II) was used for disruption of the lamp-1 structural gene in ES cells. The open reading frame of the gene is disrupted by insertion of aneo cassette in a BglII site at lamp-1exon 3 corresponding to cDNA nucleotide position 323 and amino acid 107 of 382 amino acids, respectively (8Chen J.W. Cha Y. Yuksel K.U. Gracy R.W. August J.T. J. Biol. Chem. 1988; 263: 8754-8758Abstract Full Text PDF PubMed Google Scholar). The targeting construct was introduced into E-14-1 ES cells (45Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (925) Google Scholar), and G418- and gancyclovir-resistant colonies were analyzed by Southern blotting. Using the 3′ external probe (Fig. 1 A), in two out of 96 independent clones tested an additional HindIII DNA restriction fragment was detected, indicating a homologous recombination in one of the lamp-1 gene alleles (Fig.1 B). These results were confirmed by hybridizingBglII-digested DNA with the 5′ external probe (data not shown). The targeted ES cell clones were microinjected into C57BL/6J blastocysts, and five chimeric males were generated. Only the ES cells from ES cell clone EL20 transmitted the mutated allele through the germ line. Heterozygous offspring was identified by hybridization ofHindIII-digested DNA with the 3′ external probe (data not shown). Heterozygotes exhibit a normal phenotype and normal fertility (data not shown). Genotyping of 98 offspring from heterozygote crosses (Fig.1 C) revealed a frequency of 27.2% for homozygous mutant mice (LAMP-1 −/−), resembling the expected Mendelian frequency (25%). Hence, disruption of the lamp-1 gene does not result in embryonic lethality. To test for expression of thelamp-1 gene in LAMP-1 −/− mice, Northern blot analyses were performed. A single lamp-1-specific transcript was detectable in liver and kidney total RNA from wild-type animals, whereas no lamp-1-specific transcripts were detectable in homozygous mutant animals (Fig. 1 D). LAMP-1 protein was not detectable in brain, heart, liver, spleen, and kidney homogenates from LAMP-1 −/− animals, whereas it was readily detectable in the respective homogenates from wild-type mice (Fig.1 E). The Northern blot and Western blot analyses demonstrate that thelamp-1 gene has been inactivated and that homozygous mutant mice are devoid of LAMP-1. Homozygous mutant, heterozygous, and wild-type mice resulting from heterozygote crosses did not exhibit differences in growth and weight development (data not shown). LAMP-1-deficient mice were fertile and did not show an elevated mortality up to an age of 19 months. X-ray analyses and determination of clinical blood and serum parameters did not reveal abnormalities (data not shown). Macroscopically, no differences were observed in shape and size of individual organs prepared from knockout mice as compared with controls. Light microscopic investigation of liver, kidney, lung, spleen, and brain did not reveal any apparent change in tissue structure. Likewise, density and distribution of macrophages and microvascular architecture taken as indicators for tissue damage were unchanged. In the brain (and there most notably within the dorsal neocortical region), the normally high neuronal expression of cathepsin-D (Fig. 2 A) was replaced by a more irregular distribution pattern with a still high immunoreactivity in superficial laminae II and deep lamina VI, but now being irregularly reduced in the more central strata (Fig.2 B). It should be pointed out that this does not correlate with neuronal cell degeneration, but it could be associated with an early disturbance of lysosomal metabolism in a subpopulation of cells. In addition, astrogliosis was observed in circumscribed dorsal cortical areas spanning about 500–800 μm (Fig. 2 D; control shown in Fig. 2 C), which overlapped only partially with the regions featuring alterations in their cathepsin-D" @default.
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