From Zentrum Biochemie und Molekulare Zellbiologie,
Abteilung Biochemie II, Universität Göttingen, 37073 Göttingen, Germany, the § Institute of Biotechnology
and Electron Microscopy, University of Helsinki, FIN-00014 Helsinki,
Finland, and
Anatomisches Institut der Universität Kiel,
Otto-Hahn-Platz 8, 24043 Kiel, Germany
![]() |
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 of
lamp-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 (1), transport of degradation products from the lysosomal lumen
to the cytoplasm, and regulation of fusion and fission events between
lysosomes themselves and other organelles (2, 3).
The lysosomal membranes contain several highly
N-glycosylated proteins among which the best known are
LAMP-1 and LAMP-2. These two glycoproteins are structurally similar and
evolutionary related (4). Alignment data suggest that chicken
lysosome-endosome-plasma membrane 100 (5), rat lysosomal membrane
glycoprotein 120 (6) (also described as rat lysosomal membrane
glycoprotein 107 (7)), mouse lysosomal-associated membrane protein-1
(8), and human lysosomal-associated membrane protein A (9) also
described as human lysosomal-associated membrane protein-1 (10) are
species specific versions of the same protein designated as LAMP-1 or lysosomal membrane glycoprotein A (4, 11).
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 (9). The latter contains a Gly-Tyr motif critical for transport
to lysosomes (12, 13).
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- and
O-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 (14, 15) and metastatic potential (16-19) of
tumor cells.
The lamp-1 gene is ubiquitously expressed (20) with somewhat
higher levels in spleen, liver, and kidney (20-22). In P388 macrophages, LAMP-1 comprises about 0.1% of total cell protein, corresponding to about 2 × 106 LAMP-1 molecules/cell
(23). 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 (3, 4).
Although LAMP-1 is distributed within the cell primarily in the
lysosome, under certain circumstances, e.g. after platelet activation (24), during granulocytic differentiation and activation (14, 25), and on cytotoxic T lymphocytes (26) it is also found at the
cell surface. LAMP-1 was also found on the cell surface of highly
metastatic tumor cells (3, 16-19). It was suggested that cell
surface-expressed LAMP can serve as ligand for selectins (21, 27) and
mediate cell-cell adhesion/recognition events (28).
At present there are hardly any data as to the in vivo
functions 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 regulated in vivo.
Isolation of a Genomic Clone and Targeting Vector
Construction--
An EMBL3-129SV mouse phage library from Stratagene
Inc. (La Jolla, CA) was screened with a
550-bp1 genomic amplification
product of mouse lamp-1 corresponding to cDNA positions
118-350 (8). The probe contained exon 2 and exon 3 of mouse
lamp-1 interrupted by a small intron. The isolated mouse
lamp-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. 1A,
II) was subcloned into the plasmid vector pBluescript SKII+
(Stratagene). The neo expression cassette from pMC1neopA
(Ref. 29; 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.
8). The insertion of the neo cassette introduces a premature
translational stop codon into the open reading frame of the
lamp-1 gene. Additionally, for negative selection with
gancyclovir, a thymidine kinase cassette was inserted at the 3' site of
the KpnI fragment.
Selection of Targeted Embryonic Stem (ES) Cells and Generation of
Mutant Mice--
The targeting vector was linearized with
XhoI and introduced into the ES cell line E14-1 by
electroporation. ES cells were cultured as described by Köster
et al. (30). G418- and gancyclovir-resistant colonies were
screened by Southern blot analysis of DNA digested with
HindIII and hybridized with the 3' probe (Fig.
1A). Two ES cell clones with homologous recombination were
confirmed by digesting DNA with BglII and hybridization with
the 5' probe (Fig. 1A). The mutated ES lines were
microinjected into blastocysts of C57BL/6J mice. Chimeric males were
mated to C57BL/6J females. Mice were genotyped for the
lamp-1 gene mutation by Southern blot analysis of
HindIII-digested genomic DNA, using the 3' probe or by PCR analyses using a neomycin-specific PCR (31) 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).
Northern Blot Analysis--
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. (32). Filters
were hybridized with a lamp-1 5' probe used for genomic
library screening, a lamp-2 cDNA probe (33), and a
280-bp cDNA fragment from glyceraldehyde-3-phosphate dehydrogenase
(34). Hybridization and washing of filters were performed as described
by Lehmann et al. (35).
Western Blot Analyses--
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 (36),
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 mM
PBS, 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 (37) 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).
Immunofluorescence--
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 (38), 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 (37). 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. (39).
Subcellular Fractionation on Percoll Gradient--
Tissue
homogenates of liver, kidney, and fibroblasts were prepared in 0.25 M 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 (40) and centrifuged for 30 min at 20,000 rpm in
the vertical rotor VTi 65.1 (Beckman). Density and Osmotic Stability--
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 M
sucrose 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 Lysosomal Enzyme Assays--
Lysosomal enzymes were detected
using fluorimetric assays as described by Köster et
al. (42). Arylsulfatase A was measured using
p-nitrocatechol sulfate as substrate (43).
Metabolic Labeling of Cells and Immunoprecipitation of
Cathepsin-D and LAMP2--
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 (44) with antisera specific for mouse cathepsin-D (38).
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.
Histology--
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 0.1 M 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 (38); 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.
Immunocytochemistry of Kidney--
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
Targeted Disruption of the lamp-1 Gene and Generation of Deficient
Mice--
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. 1A,
I; for details see "Experimental Procedures"). The
targeting vector (Fig. 1A, 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 a
neo cassette in a BglII site at lamp-1
exon 3 corresponding to cDNA nucleotide position 323 and amino acid
107 of 382 amino acids, respectively (8).
The targeting construct was introduced into E-14-1 ES cells (45), and
G418- and gancyclovir-resistant colonies were analyzed by Southern
blotting. Using the 3' external probe (Fig. 1A), 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.
1B). These results were confirmed by hybridizing
BglII-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 of
HindIII-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.
1C) revealed a frequency of 27.2% for homozygous mutant
mice (LAMP-1 Inactivation of the lamp-1 Gene--
To test for expression of the
lamp-1 gene in LAMP-1
LAMP-1 protein was not detectable in brain, heart, liver, spleen, and
kidney homogenates from LAMP-1
The Northern blot and Western blot analyses demonstrate that the
lamp-1 gene has been inactivated and that homozygous mutant mice are devoid of LAMP-1.
Phenotype of LAMP-1-deficient Mice--
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. 2A) 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.
2B). 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. 2D; control shown
in Fig. 2C), which overlapped only partially with the
regions featuring alterations in their cathepsin-D expression.
Normal Lysosomal Morphology and Function--
Ultrastructural
analysis of cell types known to be rich in lysosomes such as Kupffer
cells (Fig. 3, A and
B), cultured embryonic fibroblasts (Fig. 5, compare
A and B), and kidney-proximal tubule epithelial
cells (Fig. 5, C and D) did not reveal
abnormalities in size, distribution, and shape of the lysosomal
compartment in LAMP-1-deficient cells. These observations were also
confirmed in immunofluorescence analyses of peritoneal macrophages,
embryonic fibroblasts (not shown), and adult fibroblasts. Neither
staining with antibodies against the lysosomal proteinase cathepsin-D
nor with antibodies against the lysosomal membrane proteins LAMP-2 and
LIMP-2 revealed differences between LAMP-1-deficient and control mice
(shown for LAMP-2 in Fig. 3, C and D). To
investigate if changes of lysosomal milieu have occurred affecting
lysosomal hydrolases, the specific activities of five lysosomal enzymes were determined in brain, heart, liver, spleen, kidney, and serum of
age- and sex-matched LAMP-1-deficient and control mice. No significant
differences (four animals of each genotype were analyzed) were observed
in specific enzyme activities of arylsulfatase A,
Lysosome-enriched fractions of LAMP-1-deficient and control kidneys
were used to examine (two independent experiments) the osmotic
stability of lysosomes during an incubation for up to 20 min in 0.25 M glucose. Whereas lysosomes are stable at 0.25 M sucrose, they rapidly lyse during incubation in glucose
due to import of glucose and osmotic swelling (46). No changes in fragility and integrity of lysosomes were observed in both genotypes (Fig. 3F). Even small differences in lysosomal pH are known
to alter the processing of cathepsin-D (47). The processing of cathepsin-D was normal in LAMP-1-deficient fibroblasts from adult (Fig.
3G) and embryos of day 12 (not shown). Also the sorting of
newly synthesized cathepsin-D to lysosomes was not affected by LAMP-1
deficiency (Fig. 3G).
Lysosomes were labeled by feeding BSA-gold to cultured LAMP-1-deficient
and control embryonic fibroblasts for 2 h. BSA-gold-containing lysosomal structures could easily be identified in both genotypes. The
morphology of the lysosomal compartment and the amount of BSA-gold
found inside these organelles were similar in both kinds of fibroblasts
(Fig. 5, A and B). Immunoelectron microscopy of LAMP-2 in control and LAMP-1-deficient kidney-proximal tubule cells
further showed the normal size and morphology of lysosomes in the
deficient cells (Fig. 5, B and C).
LAMP-2 Up-regulation in LAMP-1-deficient and Heterozygote
Tissues--
Since no obvious alterations of several lysosomal
parameters were found, we examined whether the loss of LAMP-1 leads to
a compensatory increase of other lysosomal membrane proteins. Western blot analyses of several tissues of LAMP-1-deficient mice revealed an
increased immunoreactivity of LAMP-2 (Fig.
4A). The increase was most
pronounced in kidney, but also clearly detectable in spleen and heart
(Fig. 4A). The amount of LAMP-2 in LAMP-1-deficient liver
appears to be the same as in control liver. Probing of the same
membranes with an anti-LIMP-2 (37) antibody showed only moderate
changes in the level of this lysosomal membrane protein (Fig.
4A). Whereas the amount of LIMP-2 appears to be reduced in
LAMP-1 deficient brain and spleen, the amounts are equivalent in heart
and liver but slightly increased in LAMP-1-deficient kidney. It is
notable that there appears to be a slight reduction of the LIMP-2
molecular weight in LAMP-1-deficient tissues (Fig. 4A).
Analyses of kidney extracts from three age- and sex-matched control and
LAMP-1-deficient mice confirmed the increased immunoreactivity of
LAMP-2 in LAMP-1-deficient kidneys (Fig. 4B). LIMP-2
expression was variable between the genotypes, i.e. slightly
elevated in two LAMP-1-deficient kidneys but decreased in the third
LAMP-1-deficient kidney extract (Fig. 4B). This analysis was
extended to kidneys of heterozygotes (Fig. 4C). Loss of one
lamp-1 allele decreases expression of LAMP-1, while
expression of LAMP-2 is increased. Again, expression of LIMP-2 is not
affected or is only moderately affected (Fig. 4C).
Densitometric evaluation of Western blot experiments revealed that
LAMP-2 was increased 2.7 ± 0.8-fold in LAMP-1-deficient (n = 6) and 1.7 ± 0.4-fold in LAMP-1 heterozygote
(n = 3) kidneys (Fig. 4D).
Immunogold labeling for LAMP-2 in kidney lysosomes of LAMP-1-deficient
and control animals encountered in the proximal tubuli revealed clear
membrane staining and indicated that there is a somewhat higher amount
of gold particles in LAMP-1-deficient lysosomal membranes (Fig.
5, C and D).
Immunofluorescence analyses of kidney sections confirmed the increased
LAMP-2 immunoreactivity in LAMP-1-deficient kidneys (Fig. 5,
E and D). As expected, LAMP-1 immunoreactivity was absent in LAMP-1-deficient kidneys (inset in Fig.
5F), whereas it could be easily identified in control
kidneys (inset in Fig. 5E). Immunoreactivity for
the lysosomal proteinase cathepsin-D was not different in control (Fig.
5G) and LAMP-1-deficient (Fig. 5H) kidneys. This
suggests that the total number of lysosomal vesicles is not affected by
the LAMP-1 deficiency.
Normal lamp-2 Expression and LAMP-2 Stability in LAMP-1-deficient
Tissues--
To see whether the up-regulation of LAMP-2 correlates
with an increased expression of lamp-2 Northern blot
analysis of kidney, brain, heart, and liver (shown for kidney in Fig.
6A) were performed. In none of
these tissues were lamp-2 transcripts significantly increased in LAMP-1-deficient mice.
To determine whether an increased half-life of LAMP-2 may cause the
increase of LAMP-2, fibroblasts from adult mice were metabolically labeled with [35S]methionine and then chased for up to
96 h. The LAMP-2 immunoprecipitates (Fig. 6B) were
quantified by densitometry (Fig. 6C). It became apparent
that the stability of LAMP-2 is not affected by LAMP-1 deficiency. It
should be noted, however, that in fibroblasts from adult
LAMP-1-deficient mice, the level of LAMP-2 is only 1.5 times elevated
compared with controls (not shown).
LAMP-1 and LAMP-2 are the major components of the lysosomal
membrane. Estimates based on immunopurification procedures calculated that both proteins account for about 50% of lysosomal membrane proteins (48) and 0.1% of total cell protein (23). Although much is
known about the structure and lysosomal trafficking of LAMP-1 and
LAMP-2 (9, 12, 13), the proposals for their physiological function are
only of a hypothetical nature. The lysosomal membrane plays a vital
role in the proper function of lysosomes by sequestering numerous acid
hydrolases from the rest of the cytoplasmic components. Moreover, it is
likely to be involved in maintaining an acidic intralysosomal
environment, transport of amino acids, and mono- and oligosaccharides,
resistance to degradation by lysosomal hydrolases and its ability to
interact and fuse with other membrane organelles, including endosomes, phagosomes, and plasma membranes (3, 4). In order to better understand
the possible contribution of one of the major components of the
lysosomal membrane, we have generated mice that are deficient for
LAMP-1.
Despite its abundance in the lysosomal membrane, the deficiency of
LAMP-1 is apparently well tolerated in mice. If LAMP-1 is critical for
lysosomal integrity, lysosomal pH, and lysosomal catabolism, we
reasoned that a deficiency of LAMP-1 would cause alterations of the
morphology, number, distribution, and stability of lysosomes as well as
reduced lysosomal hydrolase activities and altered processing of
lysosomal enzymes. For this reason we have first investigated possible
alterations of the lysosomal compartment by morphological and
functional analyses. Immunofluorescence using lysosomal markers,
subcellular fractionation, activity determination of lysosomal enzymes,
and Western blot as well as processing and secretion analysis of
cathepsin-D, glucose loading experiments, and two-dimensional gel
electrophoresis of fractions enriched in lysosomal membrane proteins
(data not shown) failed to demonstrate specific differences between
LAMP-1-deficient and control cells. The negative outcome of these
experimental approaches suggested that LAMP-1 is either dispensable or
that the loss of LAMP-1 is compensated by another protein or other
proteins. Since the latter appeared to be the more likely possibility,
two other lysosomal membrane proteins were quantified using immunoblot analyses.
These experiments clearly demonstrated that the structurally related
LAMP-2 protein is up-regulated in the majority of LAMP-1-deficient tissues tested. The specificity of this up-regulation was examined by
probing the same blots for LIMP-2, another component of the lysosomal
membrane. In contrast to LAMP-2, the immunoreactivity for LIMP-2 was
only moderately changed, and its expression pattern in LAMP-1-deficient
tissues appeared to vary from mouse to mouse. However, we cannot
completely exclude the possibility that also LIMP-2 and/or other
lysosomal membrane proteins (e.g. LIMP-1/CD63, macrosialin/CD68) can contribute to the compensation of the LAMP-1 deficiency. In kidney homogenates, LAMP-2 was about 2.7-fold higher than in controls. In homogenates from heterozygotes, LAMP-2 was 1.7-fold higher, while LAMP-1 was reduced to about half. This indicates
that loss of a lamp-1 allele induces an elevation of LAMP-2,
while expression of the remaining lamp-1 allele is
apparently not affected. The increased LAMP-2 expression was also
confirmed in LAMP-2 immunofluorescence analyses of kidney sections.
The weak phenotype of LAMP-1-deficient mice suggests that the increased
LAMP-2 can efficiently compensate for the loss of LAMP-1. The LAMP-2
up-regulation is not due to an increased expression of the
lamp-2 gene or stability of lamp-2 transcripts.
Either mechanism would have caused an increased amount of
lamp-2 mRNA detectable in Northern blot (49). An
increased half-life of LAMP-1 has been correlated with an increased
activity of the enzyme Of interest is the astrogliosis and altered cathepsin-D distribution in
LAMP-1-deficient brains. Since almost no LAMP-2 is detectable in mouse
brain (Fig. 3A), one might speculate that brain is a tissue
where LAMP-1 deficiency cannot be compensated for by an increase of
LAMP-2 and hence develop the observed alterations. In mice made
deficient for the lysosomal acid phosphatase, similar alterations were
observed and were thought to be early markers for lysosomal alterations
(50). However, future work to determine the nature and extent of these
lesions is in progress.
Taken together, this study demonstrates that the morphology and
function of lysosomes appears to be normal in LAMP-1-deficient mice and
that the deficiency of LAMP-1 neither produces an overt phenotype nor
affects viability and fertility of mice. The up-regulation of the
structurally related LAMP-2 suggests a functional overlap between both
LAMP molecules. These overlapping functions are supported by the
phenotype of LAMP-1/LAMP-2 double deficient mice. Whereas the single
deficient mice are fertile and viable, the loss of both LAMP-molecules
leads to embryonic
lethality.2
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hexosaminidase
activity were determined in each of 12 fractions collected. Fractions
corresponding to dense vesicles were pooled according to the
distribution of
-hexosaminidase activity and centrifuged for 1 h at 100,000 × g. The membraneous layer
(lysosome-enriched pellet) floating above the pelleted silica was
collected (41).
20 °C until
-hexosaminidase enzyme activity measurements. To calculate the total
-hexosaminidase activity (100% of the possible
-hexosaminidase enzyme activity), one sample was incubated in 0.25 M
sucrose in the presence of 1% Triton X-100.
-hexosaminidase
activities in the supernatants were calculated as percentages of total activity.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (61K):
[in a new window]
Fig. 1.
Targeted disruption of the lamp-1
gene. A, strategy for inactivation of the
lamp-1 gene by homologous recombination in ES cells.
I, partial structure of the genomic locus representing about
13 kbp of the lamp-1 gene region. Exons are indicated by
open boxes, and flanking introns are indicated by
solid lines. Bars labeled
5' and 3' denote DNA probes used for Southern
blot analysis. II, targeting vector pCK-Lamp-1(neo) with
5.3-kbp homology to the lamp-1 gene locus. The
neo cassette was inserted into a BglII
restriction site in exon 3. The arrow marks the direction of
transcription of the neo gene and thymidine kinase cassette.
III, predicted lamp-1 gene locus after homologous
recombination. B, Southern blot analysis of ES cell clones.
The 3' probe was hybridized to HindIII-digested genomic DNA
from ES cell clones (EL-18, EL-19, and EL-20). An additional 5.3-kbp
DNA fragment indicates a targeted allele. C, PCR analysis of
tail-genomic DNA with an exon-specific PCR amplifying a 0.6-kb fragment
in +/+, a 0.6- and a 1.8-kb fragment in +/ , and a 1.8-kb fragment in
/
mice, respectively. A neo cassette-specific PCR
amplifies a 0.4-kb fragment in +/
and
/
mice, respectively.
D, Northern blot analysis of lamp-1 expression.
Total RNA (10 µg) was hybridized using a lamp-1 genomic
probe containing exons 2 and 3 and a murine glyceraldehyde-3-phosphate
dehydrogenase probe. E, Western blot analysis of LAMP-1
expression using a hybridoma supernatant against mouse LAMP-1 (1D4B;
Developmental Studies Hybridoma Bank). In +/+ tissue extracts, the
glycosylated LAMP-1 molecules were detected. In
/
tissues, no
LAMP-1 product was found.
/
), resembling the expected Mendelian frequency
(25%). Hence, disruption of the lamp-1 gene does not result
in embryonic lethality.
/
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. 1D).
/
animals, whereas it was readily
detectable in the respective homogenates from wild-type mice (Fig.
1E).
View larger version (112K):
[in a new window]
Fig. 2.
Altered cathepsin-D immunoreactivity and mild
astrogliosis in LAMP-1-deficient brains. In the neocortex of adult
LAMP-1 ( /
) mice (B), the characteristic intense
cathepsin-D immunoreactivity of neurons (A) is irregularly
reduced. Relatively normal patterns of immunoreactivity are confined to
superficial lamina 2, some cells of lamina V, and lamina VIb. The
neocortex of these animals also features a regional astrogliosis,
i.e. areas of a diameter of several hundred µm, spanning
about
along the radial dimension, that exhibit a
considerable increase in glial fibrillary acidic protein-positive
astrocytes (D). Glial fibrillary acidic protein reaction is
weak in control neocortex (C). The bars represent
200 µm (A and B) or 50 µm (C and
D).
-galactosidase,
-glucuronidase,
-hexosaminidase, and
-mannosidase (Fig.
3E). In addition, Western blot analysis of major organs also
revealed that the concentration of the lysosomal proteinase cathepsin-D
was not different from controls (data not shown). Homogenates of
fibroblasts, brain, liver, and kidney were subjected to subcellular
fractionation using Percoll gradients. The gradient fractions were
analyzed for density and
-hexosaminidase activity. No differences in
the
-hexosaminidase profiles were observed between LAMP-1-deficient
and control tissues, indicating that LAMP-1-deficient and control
lysosomes do not differ in shape and density (data not shown).
View larger version (78K):
[in a new window]
Fig. 3.
Normal lysosomal morphology and function in
LAMP-1-deficient mice. An electron microscopic image of Kupffer
cells derived from control (A) and LAMP-1-deficient
(B) livers is shown. Electron-dense lysosomal structures are
labeled with arrows. S, sinosidual space;
Ku, , Kupffer cell; N, nucleus. The bar
represents 1.5 µm. Inserts represent higher magnification
of lysosomal structures in control (A) and LAMP-1-deficient
(B) Kupffer cells. C and D,
immunofluorescence analyses of control (C) and
LAMP-1-deficient embryonic fibroblasts (D). Cells were
labeled with LAMP-2 and goat anti-rat Texas Red. E, specific
lysosomal enzyme activities (milliunits/mg) in control
(filled bars) and LAMP-1-deficient
(open bars) tissues and sera. The values are the
means ± S.E. (bars) from four separate experiments,
i.e. four age- and sex-matched control- and LAMP-1-deficient
animals, respectively. Each measurement was done in duplicate.
F, incubation of control (filled
symbols) and LAMP-1-deficient (open
symbols) enriched lysosomal fractions from kidneys in 0.25 M sucrose and 0.25 M glucose solutions. The
percentage of free -hexosaminidase activity released after lysosomal
breakage in supernatant after ultracentrifugation is given. Total
-hexosaminidase activity (100%) was calculated after incubation in
1% Triton X-100. Two independent experiments were performed.
G, processing and secretion of cathepsin-D in control
(filled symbols) and LAMP-1-deficient
(open symbols) adult fibroblasts. Cells were
metabolically labeled and immunoprecipitated with a
cathepsin-D-specific antiserum (38). The percentage of the intermediate
form of cathepsin-D appearing in cells (processing) and of the
precursor form secreted into the medium (secretion) was calculated
after densitometry. Two independent experiments were performed.
View larger version (48K):
[in a new window]
Fig. 4.
Increased LAMP-2 expression in
LAMP-1-deficient and LAMP-1 heterozygote tissues. A, Western
blot analysis of control and LAMP-1-deficient organs incubated with a
LAMP-2-specific hybridoma supernatant (Abl 93; Developmental Studies
Hybridoma Bank) and with a LIMP-2-specific antiserum (37). In a
parallel SDS-PAGE, the same amounts of protein extracts were applied
and stained with Coomassie Blue. B, Western blot analysis of
three different (sex- and age-matched) control and LAMP-1-deficient
kidneys incubated with a LAMP-2-specific antibody and a LIMP-2 specific
antiserum (37). C, Western blot analyses of kidney tissues
of control, LAMP-1 heterozygote (+/ ) and LAMP-1-deficient (
/
)
mice incubated with a LAMP-1-specific antibody (1D4; top), a
LAMP-2-specific antibody (Abl 93, middle), and a
LIMP-2-specific antiserum (bottom). The membrane was used
subsequently for all three antibodies. D, densitometric
evaluation of all Western blot analyses demonstrating increased LAMP-2
expression in LAMP-1 heterozygote and LAMP-1-deficient kidneys.
n = number of different kidney extracts/genotype tested
for LAMP-2 expression.
View larger version (108K):
[in a new window]
Fig. 5.
Lysosomal morphology revealed
by BSA-gold endocytosis and immunocytochemical analysis. Shown are
embryonic fibroblasts from control (+/+) (A) and
LAMP-1-deficient mice ( /
) (B) after 2-h BSA-gold
endocytosis. Lysosomal BSA-gold-containing structures are indicated by
arrows. C, immunogold labeling of LAMP-2 in
control and LAMP-1-deficient (D) kidney-proximal convoluted
tubule cells. Immunogold particles are concentrated at the lysosomal
membrane, revealing similar size and morphology of lysosomes in the
control and deficient cells. E, immunofluorescence analysis
of proximal tubules from control and LAMP-1-deficient kidneys
(F) using an antibody against LAMP-2 (Abl 93). Note the
increased immunoreactivity in LAMP-1-deficient kidneys.
Insets in E and F represent staining
with a LAMP-1-specific antibody. Cathepsin-D immunoreactivity is not
altered in control (G) and LAMP-1-deficient (H)
kidney sections. All immunofluorescence data were obtained using the
same exposure times and sections of the same thickness.
Scale bars in A and B, 580 nm; scale bars in C and D,
500 nm; scale bars in E-H, 17 µm.
View larger version (43K):
[in a new window]
Fig. 6.
Unchanged lamp-2 expression
and LAMP-2 stability in LAMP-1-deficient tissues. A,
Northern blot analyses of lamp-2 expression. Kidney
total RNA was hybridized with LAMP-2 murine cDNA
(33) and a murine glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (34) probe. Two separate experiments are shown.
B, immunoprecipitation of adult control and LAMP-1-deficient
fibroblasts with a hybridoma supernatant specific for LAMP-2 (Abl 93).
Cells were metabolically labeled and chased for up to 96 h.
C, densitometric evaluation of the immunoprecipitation
experiment described for B. Shown is the percentage of
LAMP-2 protein resistant to degradation. Closed
symbols represent values derived from control fibroblasts,
and open symbols represent values from
LAMP-2-deficient fibroblasts. The immunoprecipitation experiment shown
in B was confirmed by an independent experiment (not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
3-N-acetylglucosaminyltransferase, a key enzyme in
the synthesis of N-acetyllactosamine side chains (14). Such
an increase in LAMP-2 stability was not observed in LAMP-1-deficient
fibroblasts, where LAMP-2 was only moderately increased. LAMP-2
expression may therefore be translationally regulated.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank N. Hartelt, M. Grell, and D. Niemeyer for excellent technical assistance; K. Rajewski, (Universität Köln, Köln, Germany) for providing the E-14-1 cell line; and O. Schunck and K. Nebendahl for veterinary advice.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Sa 683/1-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported in part by the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Yamanouchi Foundation for Research on Metabolic Disorders.
** To whom correspondence and reprint requests should be addressed: Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Goßlerstraße 12D, 37073 Göttingen, Germany. Tel.: 49-551-395932; Fax: 49-551-395979; E-mail: saftig{at}uni-bc2.gwdg.de.
2 P. Saftig, unpublished data.
![]() |
ABBREVIATIONS |
---|
The 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 albumin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|