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PubMed1995

The family of LAMP-2 proteins arises by alternative splicing from a single gene: characterization of the avian LAMP-2 gene and identification of mammalian homologs of LAMP-2b and LAMP-2c.

The two lysosome-associated membrane proteins, LAMP-1 and LAMP-2, are major integral membrane proteins of the lysosomes. They also occur in the plasma membrane, where they have been discovered independently as principal lactosaminoglycan-bearing glycoproteins and as tumor antigens. Avian LAMP-2 has recently been shown to be encoded by at least three transcripts resulting in variant transmembrane and cytoplasmic domains (Hatem et al., 1995). We report isolation and characterization of chicken genomic clones indicating that the three transcripts are the result of alternative splicing of a single LAMP-2 gene. Only a single LAMP-2, homologous to chicken LAMP-2a, has been described in mammals. To ascertain whether multiple forms of LAMP-2 also occur in mammals, we cloned cDNAs encoding LAMP-2 variants homologous to avian LAMP-2b and LAMP-2c from mouse brain cDNA libraries. Thus, the family of LAMP-2 proteins is conserved from bird to mammals and the diversity is generated by alternative splicing of a single LAMP-2 gene.

Amino Acid Sequence↗

PubMed1993

The genes of major lysosomal membrane glycoproteins, lamp-1 and lamp-2. 5'-flanking sequence of lamp-2 gene and comparison of exon organization in two genes.

Human lysosomal membrane glycoproteins lamp-1 and lamp-2 are the major sialoglycoproteins present in lysosomal membranes. The expression of lamp-2 molecules is uniquely regulated, whereas lamp-1 is constitutively synthesized. In order to investigate the unique expression of lamp-2, and the gene evolution of lamp-1 and lamp-2, we isolated genomic phage clones encoding these glycoproteins. Comparison of the genomic and cDNA sequences revealed that the lamp-2 gene consists of nine exons. The transcriptional start site of the lamp-2 gene was determined by primer extension analysis. In order to locate the transcriptional regulatory region of this gene, various regions of 5'-sequences were tested for promoter activity using chloramphenicol acetyltransferase as a reporter molecule. The results revealed that the 5'-flanking sequence from -172 to -20 base pairs has strong promoter activity. In this sequence, potential SP1 and AP-1 binding sites and CAAT boxes are found. Most notably, the promoter activity is suppressed if the 5' farther upstream KpnI repeat sequence is included in the tested 5'-flanking sequence, thus suggesting that the KpnI repeat sequence may have some regulatory function in the lamp-2 gene expression. Comparison of the exon organization of human lamp-2 and lamp-1 genes, or chicken lamp-1 gene, reveals that these two proteins utilize the same exon phase in corresponding introns. Furthermore, each exon encodes almost identical portions of the proteins. On the other hand, the amino acid sequence of human lamp-1 is more homologous to lamp-1 of other species than it is to human lamp-2. These results indicate that lamp-1 and lamp-2 genes were most likely produced by duplication of a primordial gene, which took place early in evolution.

Amino Acid Sequence↗

PubMed2004

LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced secretion of human mast cells.

BACKGROUND: Mast cells are resident tissue cells that induce anaphylactic reactions by rapidly releasing mediators after antigen-mediated cross-linking of immunoglobulin E receptors. In the similarly active peripheral blood basophilic leukocyte, lysosome-associated membrane protein 3 (LAMP-3; CD63) has been described as an activation marker, but LAMPs have not been investigated in normal tissue mast cells. METHODS: Intra- and extracellular expressions of LAMP-1 (CD107a), LAMP-2 (CD107b), and LAMP-3 (CD63) were analysed by flow cytometry, immunocytochemistry, and functional assays in unstimulated and stimulated leukemic human mast cell line 1 (HMC-1) and skin mast cells. RESULTS: On flow cytometry, all mast cells expressed LAMP-3 at their cell membranes, whereas LAMP-1 and LAMP-2 were barely detectable (HMC-1 cells) or expressed at low levels (<10% of skin mast cells). After fixation and permeabilisation, high intracellular levels of all three LAMPs were noted in both cell types. After stimulation, a rapid translocation of intracellular LAMPs to the cell membrane, with an associated release of histamine, leukotriene C(4) and prostaglandin D(2), was ascertained in skin mast cells only. CONCLUSION: These results show that LAMP-1 and LAMP-2 are activation markers for normal mast cells. The lack of LAMP translocation after activation of leukemic mast cells may be related to maturation or malignancy-associated defects of these cells.

Antigens, CD↗

PubMed1988

Cloning of cDNAs encoding human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Comparison of their deduced amino acid sequences.

We have isolated and sequenced cDNA clones corresponding to the entire coding sequences of the human lysosomal membrane glycoproteins, lamp-1 and lamp-2 (h-lamp-1 and h-lamp-2). The deduced amino acid sequences indicate that h-lamp-1 and h-lamp-2 consist of 416 and 408 amino acid residues, respectively, and suggest that 27 and 28 NH2-terminal residues are cleavable signal peptides. The major portions of both h-lamp-1 and h-lamp-2 reside on the luminal side of the lysosome and are heavily glycosylated by N-glycans: h-lamp-1 and h-lamp-2 were found to contain 19 and 16 potential N-glycosylation sites, respectively. The findings are consistent with the results obtained by endo-beta-N-acetylglucosaminidase F treatment of h-lamp-1 and h-lamp-2 precursors, described in the preceding paper (Carlsson, S. R., Roth, J., Piller, F., and Fukuda, M. (1988) J. Biol. Chem. 263, 18911-18919). These N-glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and serine in h-lamp-1 or proline and threonine in h-lamp-2. The two domains of h-lamp-1 on each side of the hinge region are homologous to each other, whereas no such homology was detected between the two domains of h-lamp-2. Both proteins have one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH terminus, and contain a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end. Comparison of h-lamp-1 and h-lamp-2 sequences reveal strong homology between the two molecules, particularly in the proximity to the COOH-terminal end. It is possible that this portion is important for targeting the molecules to lysosomes. These results also suggest that lamp-1 and lamp-2 are evolutionarily related. Comparison of known lamp-1 sequences among different species, on the other hand, show that human lamp-1 has more similarity to lamp-1 from other species than to human lamp-2. This fact, taken together with the finding that h-lamp-2 lacks repeating domains, suggests that lamp-1 and lamp-2 diverged from a putative ancestor gene in early stages of evolution. These results also suggest that lamp-1 and lamp-2 probably have distinctly separate functions despite the fact that they share many structural features.

Amino Acid Sequence↗

PubMed1996

Biosynthetic transport of a major lysosome-associated membrane glycoprotein 2, lamp-2: a significant fraction of newly synthesized lamp-2 is delivered to lysosomes by way of early endosomes.

Lysosomal membranes contain two highly glycosylated proteins, designated as lamp-1 and lamp-2, as major components. Lamp-1 and lamp-2 are similar to each other in the protein structure. Here, we investigated the biosynthetic transport of lamp-2 through the endocytic vacuoles in cultured rat hepatocytes in comparison with that of lamp-1, which has previously been studied [Akasaki et al. (1995) Exp. Cell Res. 220, 464-473]. Newly synthesized lamp-2 (NS-lamp-2) was transported to the trans-Golgi from rough endoplasmic reticulum with a half time (t1/2) of 32 min, more slowly than NS-lamp-1 (t1/2 = 13 min). After leaving the trans-Golgi, NS-lamp-2 is transferred to at least three compartments; the cell surface (t1/2 = 47 min), cell peripheral early endosomes (t1/2 = 38 min) and perinuclear late endosomes (t1/2 = 48 min). NS-lamp-2 transported to any compartment is delivered finally to lysosomes (t1/2 = 90 min). A significant fraction of NS-lamp-2 (45% of the total) was transported from the trans-Golgi to early endosomes, and then delivered to dense lysosomes via perinuclear late endosomes, whereas a major portion of NS-lamp-1 follows an intracellular route to late endosomes without passing through the cell periphery. NS-lamp-2 leaves the cell peripheral region more rapidly than NS-lamp-1. The kinetic and quantitative data for biosynthetic transport of NS-lamp-2 to early endosomes and the cell surface indicate that NS-lamp-2 may be transported first to early endosomes, from which a small portion of it (approximately 3.5% of the total) moves to the plasma membrane via a recycling system. In contrast, a small fraction of NS-lamp-1 is transported to the plasma membrane directly from the trans-Golgi, since NS-lamp-1 is delivered to the plasma membrane and early endosomes with almost the same half times.

Animals↗

PubMed2002

Influences of the lysosomal associated membrane proteins (Lamp-1, Lamp-2) and Mac-2 binding protein (Mac-2-BP) on the prognosis of pancreatic carcinoma.

BACKGROUND: Lamps and Mac-2-BP are ligands of galectin-3, and they were suggested to influence tumor proliferation and metastasis formation. The authors studied the expression of Lamp-1, Lamp-2, and Mac-2-BP in pancreatic carcinoma and evaluated their influence on patient prognosis. METHODS: Northern blot analysis, in situ hybridization, and immunohistochemistry were performed in 12 normal and 28 pancreatic carcinoma tissue samples and in pancreatic carcinoma cell lines. The molecular findings in the tumor samples were correlated with the prognosis and histopathologic tumor characteristics. In addition, in Lamp-1 transfected CAPAN-1 pancreatic carcinoma cells, cell proliferation was analyzed. RESULTS: Lamp-1, Lamp-2, and Mac-2-BP were overexpressed in 61% (1.6-fold increase, not significant), 71% (3.0-fold increase, P < 0.01), and 93% (5.6-fold increase, P < 0.01) of the pancreatic carcinoma samples. Lamp-1 and Lamp-2 immunoreactivity was present at the luminal side of the ductal carcinoma cells whereas Mac-2-BP immunoreactivity was diffusely spread over the whole cytoplasm and the nucleolus of ductal carcinoma cells. Correlation of the molecular data with clinical patient parameters revealed that patients whose tumors exhibited high Lamp-1 mRNA expression lived significantly longer (median, 17 months) after tumor resection than patients whose tumors exhibited low to moderate Lamp-1 mRNA levels (median, 8 months; P < 0.02). No relation between Lamp-2 and Mac-2-BP mRNA expression and any of the histopathologic parameters was found. Lamp-1 transfected CAPAN-1 cells showed decreased cell growth compared with the nontransfected cells. CONCLUSIONS: Lamp-1 might influence local tumor progression rather than the formation of tumor metastasis in pancreatic carcinoma, whereas Mac-2-BP and Lamp-2 seem to have little influence on these parameters in pancreatic carcinoma.

Aged↗

PubMed2002

Response of psoriasis to sunbed treatment: comparison of conventional ultraviolet A lamps with new higher ultraviolet B-emitting lamps.

BACKGROUND: Sunbeds fitted with conventional ultraviolet (UV) A lamps that have about 0.7% UVB emission are widely used by patients with psoriasis even though they are minimally effective. A new fluorescent sunbed lamp has been developed that emits a higher proportion of UVB (4.6%) than conventional lamps and also requires shorter exposure times to achieve equivalent erythema. OBJECTIVES: To perform a randomized, within-patient comparison of conventional sunbed lamps (Cleo Performance) with the new lamps (Cleo Natural) in the treatment of psoriasis. METHODS: A sunbed and canopy unit were modified to allow exposure to Cleo Performance lamps on one side of the body (front and back) and Cleo Natural lamps to the other side of the body. Two studies were done. In study 1, equal erythemal doses were given from the two lamp types. In study 2, equal exposure times were given. We treated 34 patients with psoriasis, giving 12 exposures over a period of 4 weeks. Assessment was made using a modified Psoriasis Area and Severity Index (PASI) score, individual plaque assessment and patient questionnaire. RESULTS: Fourteen patients completed each study. In study 1, there was no significant difference in median improvement in half-body PASI score for the two lamp types. In study 2, there was a significant difference in PASI score improvement between the two lamps (median Cleo Performance change minus median Cleo Natural change was - 2.20; 95% confidence interval - 3.75 to - 0.65; P = 0.006). CONCLUSIONS: That no difference in response was found when equal erythemal doses were given suggests that the spectral emission of the Cleo Natural lamp is of no greater advantage for clearance of psoriasis than conventional lamps. However, the Cleo Natural lamps are more erythemally powerful, and exposure times similar to those used in conventional sunbeds result in a significant improvement of psoriasis. The risk of non-melanoma skin cancer from different patterns of exposure to Cleo Natural lamps can be estimated using established numerical models.

Adolescent↗

PubMed1990

Two human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2, are encoded by genes localized to chromosome 13q34 and chromosome Xq24-25, respectively.

We have isolated previously cDNAs encoding two related human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2 (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S.R. (1988) J. Biol. Chem. 263, 18920-18928). In the present study, we have determined the chromosomal localization of genes for h-lamp-1 and h-lamp-2. By using the method of in situ hybridization, we have localized the gene for h-lamp-1 to chromosome 13q34 and its related gene to chromosome 12p133. The hybridization of h-lamp-1 cDNA to chromosome 12p133 was observed even when probes representing different portions of h-lamp-1 cDNA were used. On ther other hand, the gene for h-lamp-2 were localized to Xq24-25 but no cross-hybridization to chromosome 12p133 was observed even though h-lamp-1 and h-lamp-2 are highly related. These results clearly indicate that human lamp-1 and lamp-2 are coded by separate genes on different chromosomes. The present results support our hypothesis that lamp-1 and lamp-2 diverged early in evolution and they have distinct functions which emerged as soon as eukaryotic cells acquired lysosomes as subcellular compartments.

Antigens, CD↗

PubMed2006

Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy.

The lysosomal membrane proteins LAMP-1 and LAMP-2 are estimated to contribute to about 50% of all proteins of the lysosome membrane. Surprisingly, mice deficient in either LAMP-1 or LAMP-2 are viable and fertile. However, mice deficient in both LAMP-1 and LAMP-2 have an embryonic lethal phenotype. These results show that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seems to have more specific functions since LAMP-2 single deficiency has more severe consequences than LAMP-1 single deficiency. Mutations in LAMP-2 gene cause a lysosomal glycogen storage disease, Danon disease, in humans. LAMP-2 deficient mice replicate the symptoms found in Danon patients including accumulation of autophagic vacuoles in heart and skeletal muscle. In embryonic fibroblasts, mutual disruption of both LAMPs is associated with an increased accumulation of autophagic vacuoles and unesterified cholesterol, while protein degradation rates are not affected. These results clearly show that the LAMP proteins fulfil functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.

Animals↗

PubMed2004

[The effects of fluorescent lamp coated with a photocatalyst film on the number of floating microorganisms or smell molecules in a room installed with the lamp].

Fluorescent lamp that is coated with a photocatalyst film, titanium dioxide (TiO2), can catalyze the microorganisms and smell producing molecules that touch the membrane. In this report, we examined the effect of this coated lamp by the number of floating microorganisms or smell molecules in a room installed with this lamp. The number of floating microorganisms was examined independently in different laboratories before and after installing the lamps in an animal facility and a vegetable processing room of a food company. We found that the number of floating microorganisms was significantly reduced after the installation of this coated lamp. To test for smell molecules, acetaldehyde was injected into a closed chamber installed with the coated lamp and the result was compared to a control where a regular fluorescent lamp was used. The acetaldehyde concentration did not change with the regular lamp but was halved after 1h with the photocatalyst coated lamp. To test the effect in real settings, questionnaires were filled out by restroom users in several different places. About 90% of those surveyed answered that there was a reduction of smell after the installation of the photocatalyst coated lamp. We concluded that the installation of this coated fluorescent lamp reduced not only the floating microorganisms but the smell in various practical situations.

Air Microbiology↗

PubMed1986

Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2.

Several properties of the lysosomal membrane glycoproteins LAMP-1 and LAMP-2 have been analysed. Each molecule was strongly associated with lysosome membranes and was extracted only in the presence of detergent. Studies of the biosynthesis and processing of the glycoproteins showed that each contained a polypeptide core of approx. 43,000 Da as identified by use of tunicamycin and endoglycosidase H. Nascent glycoproteins pulse-labelled for 5 min with [35S]methionine were approx. 92,000 Da. These precursor molecules were processed in 30 min to highly heterogeneous mature glycoproteins of approx. 110,000 Da(LAMP-1) and 105,000 Da(LAMP-2). Concomitant with the increase in apparent Mr the molecules became endoglycosidase H resistant and acquired sialic acid residues, indicating that they were converted to complex-type oligosaccharides. The final maturation of the glycoproteins was blocked by monensin. Immunohistochemical analysis of tissues from Balb/c and Beige/J mice showed that the molecules were present on many types of cells, consistent with their presence in lysosomes. The patterns of tissue expression of LAMP-1 and LAMP-2 in the two mouse strains were the same except that the intensity of staining of LAMP-2 was less than that of LAMP-1. LAMP-2, but not LAMP-1, gave a decreased immunofluorescent staining intensity in transformed HaNIH as compared with NIH/3T3 cells. The marked similarities between the LAMP proteins raise the consideration of common functions, possibly associated with the high oligosaccharide content of the molecules.

Acetylglucosaminidase↗

PubMed1988

Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan.

Two major lysosomal membrane glycoproteins with apparent Mr approximately 120,000 were purified from chronic myelogenous leukemia cells. These glycoproteins are major sialoglycoproteins containing polylactosaminoglycan and represent approximately 0.1-0.2% of total cell proteins. A monoclonal antibody specific to one of the glycoproteins and polyclonal antibodies specific to the other glycoprotein were obtained. Immunoelectron microscopic examination of HeLa cells revealed that these two glycoproteins mainly reside in lysosomes and multivesicular bodies. Immunoprecipitation experiments showed that a number of different cell lines express these glycoproteins. However, the apparent molecular weights differed between cell lines; this probably represents differences in the amount of polylactosaminoglycan expressed by each cell line. As shown in the following paper (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928) one of the glycoproteins is very homologous to that of a mouse counterpart, m-lamp-1. The human form of this glycoprotein is therefore named human lamp-1 (h-lamp-1), while the other glycoprotein, to which the monoclonal antibody was made, is called human lamp-2 (h-lamp-2). Pulse-chase labeling experiments detected that h-lamp-1 and h-lamp-2 are produced first as precursor forms of 87.5 and 84 kDa, and treatment with endo-beta-N-acetylglucosaminidase H (endo-H) or endo-beta-N-acetylglucosaminidase F (endo-F) reduced their molecular masses to 39.5 and 41.5 kDa, respectively. It was estimated that h-lamp-1 has 18 N-linked saccharides and h-lamp-2 16, based on the results of partial digestions with endo-F. These results indicate that the two lysosomal membrane glycoproteins are extensively modified by N-glycans, and some of these were found to have polylactosaminyl repeats and sialic acid. Human lamp-1 and lamp-2, therefore, serve as good models for understanding polylactosaminoglycan formation and the biosynthesis and processing of polylactosaminoglycan-containing glycoprotein.

Amino Acids↗

PubMed1998

Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells.

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E-selectin and between Lamps and galectins (endogenous-galactoside-binding lectins). The present study examined the expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell-surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo-2 (human colon-adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin-3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin-3 binding to Lamps-expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis.

Adenocarcinoma↗

PubMed1995

The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils.

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.

Antibodies, Monoclonal↗

PubMed2005

Impact of lamp shadowing and reflection on the fluence rate distribution in a multiple low-pressure UV lamp array.

Use of mathematical modeling for determination of ultraviolet (UV) fluence in disinfection reactors requires accurate knowledge of the fluence rate distribution in a multiple lamp array. A method for measuring the fluence rate among a multiple lamp array was demonstrated using spherical actinometry. A matrix of four low-pressure UV lamps in air were investigated to evaluate the potential for shadowing and reflection to impact the fluence rate within and surrounding the lamp array. Two fluence rate distribution models were tested to determine the ability to predict the fluence rate distribution measured by the actinometers. Shadowing proved to attenuate UV light. Reflection from the lamp surface added 3-9% to the fluence rate, depending upon position in the reactor. These effects, as well as the fluence rate at various points in the lamp matrix were effectively modeled using RAD-LSI and UVCalc3D fluence rate distribution models. At fluence rates above 8mWcm(-2), the actinometry measured fluence rate was lower than the modeled rate, presumably from saturation of the actinometer solution at high fluence rates (close to the lamp). With multiple lamp reactors, the impact of shadowing can significantly affect fluence rate distribution and thus the level of microbial inactivation. If shadowing is not included in fluence rate distribution models, the fluence rate will be over predicted in the shadow zone of a neighboring lamp, falsely skewing model inactivation predictions.

Disinfection↗

PubMed1998

Sorting of lysosomal membrane glycoproteins lamp-1 and lamp-2 into vesicles distinct from mannose 6-phosphate receptor/gamma-adaptin vesicles at the trans-Golgi network.

Newly synthesized lysosomal membrane glycoproteins lamp-1 and lamp-2 are primarily sorted at the trans-Golgi network (TGN) by recognition of a tyrosine-based signal sequence in their cytoplasmic tails. It is presently unclear how this signal is recognized and what type of vesicle transports lamp-1 and lamp-2. Here, we describe a method to generate transport vesicles containing lamp proteins from the TGN in vitro. The method is based on incorporation of radioactive sialic acid in glycoproteins at the TGN by incubation of membranes with tritiated CMP-sialic acid. The generation of vesicles from labeled membranes required ATP and cytosol, and was temperature-dependent and brefeldin A-sensitive. Analysis on Nycodenz gradients revealed that lamp-vesicles were distinct from vesicles containing gamma-adaptin and mannose 6-phosphate receptor (MPR). Moreover, both these types of vesicles migrated differently than vesicles containing proteins destined for the plasma membrane. The conclusion that lamps and MPRs are sorted into different vesicles was further strengthened by the finding that whereas wortmannin both in vitro and in vivo inhibited the production of gamma-adaptin/MPR-containing vesicles, this drug had no effect on the generation of lamp-vesicles and on the sorting of lamps. The results indicate that membrane proteins containing tyrosine-based motifs for sorting at the TGN are segregated from clathrin-coated vesicles containing MPRs.

Adaptor Protein Complex gamma Subunits↗

PubMed2006

Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes.

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

Animals↗

PubMed1993

Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2.

The lysosomal membrane glycoproteins lamp-1 and lamp-2 are extensively glycosylated with a variety of different carbohydrate structures of both N-linked and O-linked type. In the present paper, we report the localization of O-linked oligosaccharides exclusively to the hinge-like regions of lamp-1 and lamp-2 isolated from human chronic myelogenous leukemia cells. In both glycoproteins, the O-glycans appear in clusters. In lamp-1, Thr-171, Thr-172, Ser-179, Ser-181, and Ser-183 were fully glycosylated, whereas Ser-169 was partially glycosylated. In lamp-2, complete glycosylation was found at Ser-167, Thr-168, Thr-172, Thr-175, Thr-176, Thr-182, and Thr-183, and partial glycosylation at Ser-179 and Thr-181, and possibly also at Thr-185. The amino acid sequences of these O-glycosylation sites are consistent with the previous reports that residues at positions -1 and +3 may influence the glycosylation reaction. Circular dichroism and nuclear magnetic resonance spectroscopy was used for the structural characterization of a synthetic peptide corresponding to residues 167 to 190 of lamp-1. The results indicated that the proline-rich O-glycan acceptor region does not adopt any typical periodic structure but differs from random-coil structure. The circular dichroism spectrum of the peptide is, however, similar to that of porcine submaxillary apomucin. A significant conformational variability was observed in this region, presumably due to a slow (on the nuclear magnetic resonance time scale) cis-trans isomerization of several proline residues. These results, taken together, strongly suggest that a hinge region does not display any typical ordered structure. The presence of O-glycans thus likely protects this region from intralumenal lysosomal proteases.

Amino Acid Sequence↗