Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, has been identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anit-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromatography with 1D4B monoclonal antibody, yielding a homogeneous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the two purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [35S]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent Mr 92,000, and then converted to heterogeneous mature forms differing in average Mr from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [35S]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent molecular weight of the molecules or the differences between the various cell lines. beta-D-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.

N-glycosylation is a major post-translational modification of proteins and involved in many diseases, however, the state and role of N-glycosylation in cartilage degeneration of osteonecrosis of femoral head (ONFH) remain unclear. The aim of this study is to identify the glycoproteins of ONFH hip cartilage. Cartilage tissues were collected from nine patients with ONFH and nine individuals with traumatic femoral neck fracture. Cartilage glycoproteins were identified by glycoproteomics based on LC-MS/MS. The differentially N-glycoproteins including glycosites were identified in ONFH and controls. A total of 408 N-glycoproteins with 444 N-glycosites were identified in ONFH and control cartilage. Among them, 104 N-glycoproteins with 130 N-glycosites were significantly differential in ONFH and control cartilage, which including matrix-remodeling-associated protein 5, prolow-density lipoprotein receptor-related protein 1, clusterin and lysosome-associated membrane glycoprotein 2. Gene Ontology analysis revealed the significantly differential glycoproteins mainly belonged to protein metabolic process, single-multicellular organism process, proteolysis, biological adhesion and cell adhesion. KEGG pathway and protein-protein interaction analysis suggested that the significantly differential glycoproteins were associated with PI3K-Akt signalling pathway, ECM-receptor interaction, protein processing in the endoplasmic reticulum and N-glycan biosynthesis. This information provides substantial insight into the role of protein glycosylation in the development of cartilage degeneration of ONFH patients.

N-glycans are diversified by a panel of glycosyltransferases in the Golgi, which are supposed to modify various glycoproteins in promiscuous manners, resulting in unpredictable glycosylation profiles in general. In contrast, our previous study showed that fucosyltransferase 9 (FUT9) generates Lewis X glycotopes primarily on lysosome-associated membrane protein 1 (LAMP-1) in neural stem cells. Here, we demonstrate that a contiguous 29-amino acid sequence in the N-terminal domain of LAMP-1 is responsible for promotion of the FUT9-catalyzed Lewis X modification. Interestingly, Lewis X modification was induced on erythropoietin as a model glycoprotein both in vitro and in cells, just by attaching this sequence to its C-terminus. Based on these results, we conclude that the amino acid sequence from LAMP-1 functions as a "Lewis X code", which is deciphered by FUT9, and can be embedded into other glycoproteins to evoke a Lewis X modification, opening up new possibilities for protein engineering and cell engineering.

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.