Newly synthesized lysosomal proteins exit the trans-Golgi network in vesicles which fuse with endosomes [1]. Soluble lysosomal enzymes are tagged with mannose-6-phosphate and are sorted in the Golgi compartment by mannose-6-phosphate receptors. Lysosomal membrane proteins have sorting signals in their cytosolic domains. Early endosomes acquire lysosomal proteins and an acidic pH during their maturation to late endosomes [2]. Lysosomes can be distinguished from endosomes by the absence of mannose-6-phosphate receptors, a lower internal pH and a distinct morphology [3]. Lysosomes appear electron dense by comparison with endosomes and can be separated by density gradient centrifugation. Lysosomes may be regarded as storage organelles for degradative enzymes, with the degradation of substrates occurring mainly in hybrid organelles of late endosomes and (auto)phagosomes [4]. Endosomes deliver endocytosed molecules from the extracellular space for degradation, and also proteins provided by the ESCRT pathway. ESCRT complexes package ubiquitinated proteins into vesicles formed by inward budding of the endosomal limiting membrane. Fusion of a late endosome with lysosomes creates a hybrid organelle of intermediate density [5]. The hybrid organelle hydrolyses its cargo - macromolecules and intraluminal vesicles - and transports the small molecular products into the cytosol. The remaining lysosomal components are condensed and form new dense-core lysosomes [6].

The lysosome's degradative machinery is potentially harmful to its limiting membrane, since it is capable of degrading intact lipid membranes. The inside of the lysosomal membrane is protected from degradation, presumably by a high abundance of heavily glycosylated membrane proteins. The membrane is quickly degraded when its glycoconjugate-free outside is exposed to the lysosomal interior [7].

Lysosomes are characterized by a high abundance of heavily glycosylated membrane proteins. These proteins form a dense coat on the inside of the lysosomal membrane. The glycoprotein coat can be visualized by electron microscopy as a thin, electron translucent halo separating the membrane from the condensed core [8,9]. It can be stained by glycoprotein-specific reagents and its thickness ranges from 5 to 12 nm with an average of 8 nm [8,9]. In comparison, the glycocalyx on the cell surface is much thicker, up to several micrometers [10]. Intracellular glycoprotein membrane coats were observed in lysosomes, endosomes, autophagosomes and secretory granules [9]. They are believed to protect vesicle membranes by limiting the access of degradative factors to the lipid bilayer.

The major lysosomal membrane proteins, the homologous LAMP-1 (lgp120, CD107a) and LAMP-2 (lgp110, CD107b), are the standard markers for the lysosomal compartment [11]. They are among the most extensively glycosylated proteins with glycan chains that outweigh the protein core and that include high-molecular-weight poly-N-acetyllactosaminoglycans [12]. LAMP-1 and 2 are type-I membrane proteins of similar length and identical domain structure. The transmembrane region is followed by a short, C-terminal cytosolic tail that comprises motifs for lysosomal targeting [1]. The luminal region comprises two similar N-glycosylated domains of about 160 residues, each with two conserved disulfide bonds (Figure ​(Figure1).1). The two domains are separated by a proline-rich, O-glycosylated 'hinge' region of about 30 amino acid residues [13].

LAMP-1 and LAMP-2 are abundant proteins, representing 0.1 to 0.2% of total cell protein [14]. They are enriched in lysosomes, late endosomes and mature (auto)phagosomes. Both proteins are ubiquitous in human tissues and cell types [15]. Their expression is particularly pronounced in metabolically active cells. The LAMP-2 gene has three splice forms, LAMP-2A, -2B, -2C, which differ in the transmembrane and cytosolic regions [15]. The LAMP-1 gene encodes for a single transcript.

Considerable knowledge on LAMP function was derived from LAMP-1 and LAMP-2 gene knockout mice [16,17] and LAMP-1/2-negative cell lines [18]. Impaired fusion of phagosomes and autophagosomes with lysosomes was observed. LAMP-1 and 2 can complement each other to a large extent. Double LAMP-1/2 knockout leads to embryonic lethality. In contrast, LAMP-1 knockout mice are healthy [16]. They display upregulated levels of LAMP-2. Knockout of the LAMP-2 gene leads to elevated postnatal mortality [17]. Surviving mice have a phenotype that corresponds to Danon disease, a rare genetic condition caused by LAMP-2 deficiency [19]. Autophagosomes accumulate in several tissues of LAMP-2 negative mice. Danon disease entails muscle weakness, heart disease and mental retardation. It is associated with disturbed autophagosome maturation and extensive accumulation of autophagosomes in muscle cells.

LAMP proteins are important regulators of lysosome fusion with autophagosomes and phagosomes [20]. Macrophages lacking either LAMP-1 or LAMP-2 are capable of normal phagocytosis. However, knockout of both genes interferes with fusion of phagosomes with lysosomes [21,22]. Phagosomes lacking both LAMP-1 and 2 did not move from the periphery of the cell towards the perinuclear lysosomes [21]. In contrast to macrophages, LAMP-1 cannot compensate for lack of LAMP-2 in neutrophil phagocytes. LAMP-2 knockout mice suffer from a high prevalence of periodontitis because their neutrophils cannot keep the responsible bacteria in check [23].

LAMP-2 has been implicated in cholesterol transport from the lysosome [18,24]. It was shown that the membrane-proximal luminal domain of LAMP-2 is specifically required to rescue the cholesterol transport deficiency of LAMP-1/2 double knockout cells [24].

LAMP-1 and 2 are major components of the glycoconjugate coat on the inside of the lysosomal membrane. It was estimated that the concentration of LAMPs is sufficiently high for the formation of a nearly continuous layer on the inner surface of the lysosomal membrane [25]. It was, therefore, expected that their removal would destabilize lysosomes. However, depletion of LAMP-1 and 2 had no detectable effect on lysosome integrity. Removal of N-linked glycans with endoglycosidase H caused rapid degradation of LAMP-1 and LAMP-2, but did not destabilize lysosomes [26]. Lysosomes of LAMP-1/2 knockout cells have abnormalities, but their limiting membranes are apparently intact [18]. Other glycoproteins and glycolipids might have provided sufficient membrane protection in these studies. Lysosomal hybrid organelles with larger limiting membrane surface and lower membrane glycoprotein density might be more dependent on LAMPs for protecting their membranes.

Intracellular glycoconjugate coats are found on the membranes of secretory granules [9]. During degranulation of Natural Killer (NK) or cytotoxic T-cells, cytotoxic effectors become activated, which can potentially harm the effector cells' cell membrane. LAMP-1 is a surface marker of NK cells that have degranulated. It can protect NK cells against their own cytotoxic effectors following degranulation (André Cohnen, University of Heidelberg, Carsten Watzl, Leibniz Research Centre for Working Environment, unpublished data).

Chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective removal of damaged cytosolic proteins (reviewed in [27]). CMA effects direct transmembrane import of cytosolic proteins into the endolysosomal system. The LAMP-2 isoform LAMP-2A functions as a receptor for cytosolic proteins and also as essential component of the CMA translocation complex [28]. Cytosolic substrate proteins bind to monomers of LAMP-2A, which then multimerizes to form the complex required for substrate transmembrane import. Membrane-associated molecules of hsc70 actively disassemble LAMP-2A into monomers to initiate a new cycle of binding and translocation [28]. Expression of LAMP-2A normally declines in the liver of mice as they age. Genetic engineering for overexpression of LAMP-2A in the liver not only restored CMA, but also (macro)autophagy and proteasomal pathways to the levels observed in young animals. As a result, the age-related decline of liver function was significantly reduced [29].

The five human members of the LAMP protein family are characterized by a conserved 'LAMP domain' directly adjacent to the single transmembrane helix (Figure ​(Figure1).1). LAMP-1 and 2 are ubiquitous proteins, whereas DC-LAMP (LAMP-3) [30], BAD-LAMP (UNC-46, C20orf103) [31] and macrosialin (CD68) [32] are only expressed in specific cell types.

Dendritic cells (DCs) are professional antigen-presenting cells with the task of activating immune responses. DC-LAMP levels increase progressively during the differentiation of human DCs from hematopoietic bone marrow progenitor cells. DCs search for pathogens in tissues in contact with the external environment. They phagocytose pathogens, become activated and migrate to lymph nodes where they present pathogen-specific antigens on their cell surface using MHC class II molecules. DC-LAMP levels rises steeply upon activation of human DCs [30]. The protein co-localizes with MHC class II molecules in an intracellular compartment. DC-LAMP is a highly specific marker for mature DCs in humans and other mammals including cattle and pigs, but it is not expressed in DCs of mice [33].

Mammals, including mice, have a second cell type with high DC-LAMP expression: type II pneumocytes [34]. These cells secrete the lung's surfactant and, like DCs, can present antigens of pathogens on MHC class II molecules [35,36]. DC-LAMP co-localizes with MHC class II molecules on the limiting membrane of surfactant protein-containing organelles [34]. In chicken, DC-LAMP expression is upregulated upon DC activation [37]. In contrast to mammals, DC-LAMP was found in nearly all chicken tissues tested.

The structure explains the complete conservation of the pair of disulfide bonds in the LAMP protein family. The first disulfide bond stabilizes the β-prism, while the second disulfide bond fixes the domain's C-terminus to the center of the kinked β-sheet. The center of the sheet is thereby linked to the transmembrane helix. Loop L3, the flexible loop L1 and the adjacent β-hairpin point sideways. In this arrangement, the loops would be in a suitable position for interacting with luminal domains of neighboring membrane proteins.

The LAMP family is characterized by a conserved, membrane-proximal domain. Sequence conservation is not restricted to this domain, but includes the adjacent transmembrane helices (Figure ​(Figure1B).1B). All human LAMPs contain a conserved proline and two conserved glycine residues in the transmembrane helix. Glycine residues mediate interactions of transmembrane helices in membrane proteins [49]. In the course of chaperone-mediated autophagy (CMA), LAMP-2A oligomerizes into multimeric complexes. Mutation of the conserved glycines in the LAMP-2A transmembrane region to alanines inhibits oligomerization and significantly reduces CMA activity [28]. The lysosomal polypeptide transporter TAPL (ABCB9) has recently been identified as a specific binding partner of LAMP-1 and LAMP-2 [50]. The interaction appears to be mediated by transmembrane regions: the binding site for the LAMPs is localized in the N-terminal domain of TAPL, which comprises four transmembrane helices but no globular domains. The LAMP-2B isoform is recognized exclusively by TAPL, which differs from LAMP-2A only in the transmembrane region and the cytosolic tail. The structure solved in this study identified a possible protein binding site (Figure ​(Figure5B).5B). It is possible that LAMPs self-oligomerize or recognize other lysosomal membrane proteins by simultaneous interactions with their luminal and transmembrane regions. The LAMP family member unc-46 (BAD-LAMP) and the vesicular GABA transporter depend on each other for their vesicular sorting [51]. This phenomenon might be due to molecular interaction of the two proteins.

The dimensions of the in silico models are consistent with literature data on the thickness of the glycoprotein coat on the lysosomal membrane (Figure ​(Figure6)6) and the density of LAMP molecules on the lysosomal membrane. In BHK cells, the surface area of lysosomal and LAMP-1 positive prelysosomal membranes was determined as 370 μm²/cell [52]. The average volume of the cells was 1,400 μm³ [53]. LAMP-1 represents approximately 0.1% of the total cellular protein content of 200 to 250 mg/ml [25]. This amounts to a density of LAMP-1 in lysosomal membranes of approximately 2,600 molecules/μm². The footprint of the LAMP-1 model in Figure ​Figure66 is about 120 nm², which would implicate that about one third of lysosomal membranes is covered by LAMP-1. The model thus supports the concept of a continuous glycoprotein layer formed by LAMP-1, the similar LAMP-2 and other lysosomal proteins.

The luminal regions of lysosomal membrane proteins are typically small and do not contain more than a single folded domain. A comprehensive list of 45 lysosomal membrane proteins [54] was analysed with PFAM. Multidomain proteins were not found in this list and immunoglobulin or fibronectin domains, which are frequent in extracellular multidomain regions, were not present. This result corresponds to the thickness of the lysosomal glycoprotein coat of 8 nm on average, which is low in comparison to the glycocalyces on cell membranes of several hundred nanometers thickness [55].