The polylactosaminoglycans of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Localization on the peptide backbones

Lysosome membrane glycoproteins, lamp-1 and lamp-2, have been shown to contain 18 and 16 N-glycans, some of which are modified by poly-N-acetyl-lactosamine. We have localized the polylactosaminoglycans to specific sites on lamp-1 and lamp-2 purified from human chronic myelogenous leukemia cells. Polylactosaminoglycan-containing glycopeptides, obtained by trypsin, pepsin, and V8 protease digestion of the glycoproteins, were isolated by Datura stramonium agglutinin affinity chromatography, gel filtration, and reverse phase high performance liquid chromatography. The poly-N-acetyllactosaminyl structures of isolated glycopeptides were confirmed by the susceptibility of their released oligosaccharides to endo-beta-galactosidase. Amino acid analysis and sequencing demonstrated that polylactosaminoglycans were located at Asn-34, Asn-93 and/or Asn-102, and Asn-195 and/or Asn-200 in lamp-1, and at Asn-4 and/or Asn-10, and Asn-279 in lamp-2. These results indicated that only certain glycosylation sites can be selectively modified by poly-N-acetyllactosamine, and those sites may confer the requirement by beta 1----3-N-acetylglucosaminyl transferase.     

Purification and characterization of human lysosomal membrane glycoproteins

Two human cell lysosomal membrane glycoproteins of approximately 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing approximately 0.05% of the total detergent-extractable protein. Both molecules were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was approximately 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membrane of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.     

Identification of two lysosomal membrane glycoproteins

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.     

The cDNA sequence of mouse LAMP-2. Evidence for two classes of lysosomal membrane glycoproteins

We describe the isolation and sequencing of a cDNA encoding the mouse lysosomal membrane glycoprotein mLAMP-2 and the sequence differences that distinguish this molecule from the LAMP-1 class of proteins. An oligonucleotide probe corresponding to the NH2-terminal amino acid sequence of purified mLAMP-2 was synthesized by the polymerase chain reaction and used to screen several cDNA libraries. cDNA clones with an insert of 1,700 nucleotides were identified and sequenced. The deduced amino acid sequence of mLAMP-2 comprises a signal sequence of 25 residues and a 390-amino acid polypeptide (Mr 43,017) with the following putative domains: a large intraluminal region (residues 1-354) with 17 N-linked glycosylation sites (Asn-X-Ser/Thr), a hydrophobic transmembrane-spanning region of 24 residues (355-378), and a COOH-terminal cytoplasmic tail of 12 residues (379-390). When this sequence is compared with those of other lysosomal membrane glycoproteins, it is apparent that mouse LAMP-2 and human LAMP-2 form one homology class (LAMP-2) that is separated from the LAMP-1 class of proteins. The sequence differences in these two classes provide a basis for comparing the structure of the proteins with their biochemical and biological properties.     

Deficiency of two human leukocyte surface membrane glycoproteins (Mo1 and LFA-1)

Structural and functional features of a novel disorder characterized by recurrent bacterial infections are reviewed. This disease is associated with a number of phagocyte adhesion defects. In 10 patients, phenotypic analysis with monoclonal antibodies (MAb) revealed the same basic defect in all patients: deficiency of at least two leukocyte surface glycoproteins, Mo1 and LFA-1. These two antigens have distinct alpha subunits (Mo1 alpha = 155 kilodaltons, LFA-1 alpha = 177 kilodaltons) noncovalently linked to a common beta subunit (94 kilodaltons). Mo1 is closely associated with or identical to a receptor for the iC3b fragment of the third component of complement. LFA-1 is involved in lymphocyte proliferation, cytotoxicity, and natural killing. MAb directed to this family of glycoproteins induce functional defects in normal cells similar to those observed in deficient cells. In normal cells, the surface expression of these glycoproteins is regulated by the state of cell activation. Mitogens and alloantigens significantly increase the surface expression of LFA-1 on T lymphocytes. Stimuli that induce degranulation in neutrophils increase the surface expression of Mo1. In all patients with combined Mo1, LFA-1 deficiency, the predominant clinical manifestations were more characteristic of a phagocyte than a lymphocyte disorder. In vitro studies, however, reveal significant defects in phytohemagglutinin-induced proliferation that are more apparent at lower concentrations of the lectin. In some families, more than one sibling is affected. Intermediate levels of Mo1 were observed on granulocytes from both parents of one child. In one family, however, only the mother had significantly reduced levels of Mo1, which indicates heterogeneity in the inheritance of this disorder.     

Monoclonal antibodies recognizing epitopes carried on both glycolipids and glycoproteins of the human milk fat globule membrane.

The molecules of the human milk fat globule membrane (MFGM) which bind four murine monoclonal antibodies (LICR LON M3, M8, M18 and M24) raised against the human MFGM have been identified. By using 'Western' blotting [Burnette (1981) Anal. Biochem. 112, 195-203] it was shown that each antibody reacted with a different set of proteins. M3 and M24 were similar in their pattern of reaction with the membrane proteins, but were quite distinct from M8 and M18, which also differed from each other. Glycopeptides prepared from the MFGM by exhaustive Pronase digestion were able to inhibit partially the binding of M3 and M24, and prevent totally the binding of M8 and M18, to the MFGM in an enzyme-linked immunoabsorbent assay. Oligosaccharides obtained by the deproteination of human milk also completely inhibited the binding of M3, M18 and M24 to the MFGM. However, the binding of M8 was not inhibited by these saccharides, and therefore M8 may not be recognizing a simple carbohydrate determinant. By using an enzyme-linked assay, M8 and M18 were shown not to bind to MFGM glycolipid, whereas M3 and M24 did, and this was confirmed by overlaying thin layer chromatograms of MFGM lipids with these antibodies. Both M3 and M24 showed a similar complex pattern of reaction, binding to more than one glycolipid moiety. By these means all four antibodies have been shown to react with antigens which involve carbohydrate side chains carried on different proteins, and two were also shown to react with such determinants on glycolipids.     

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts

 Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts. Accepted: 9 December 1998     

Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials.

Changes in the glycosylation of asparagine-linked oligosaccharides have been shown in various tumor cells, including human colon cancer. Attempts were made to elucidate the difference in Asn-linked oligo-saccharides attached to lysosomal membrane glycoproteins isolated from sublines of human colon carcinoma exhibiting high and low metastatic potentials in nude mice. Lysosomal membrane glycoproteins (lamp) 1 and 2 were immunoprecipitated from the cells after labeling with radioactive sugars, and the glycopeptides prepared were fractionated by serial lectin affinity chromatography employing immobilized concanavalin A, Datura stramonium agglutinin, and tomato lectin. Comparison of Asn-linked oligosaccharides from the different colonic carcinoma cells revealed the following features. First, the highly metastatic carcinoma cells express more poly-N-acetyllactosaminyl side chains with branched galactose residues than cells with low metastatic potential. Second, sialylation is more significant in the highly metastatic carcinoma cells than in the poorly metastatic ones. Conversely, N-acetyllactosamine units are less fucosylated in the highly metastatic cells than in poorly metastatic cells. These structural changes were apparently caused by the increase in sialyltransferase and the decrease in alpha 1----3 fucosyltransferase in the highly metastatic cells. The results also suggest that highly metastatic carcinoma cells express more sialyl Lex structures at the termini of poly-N-acetyllactosaminyl side chains than poorly metastatic carcinoma cells. Further, highly metastatic cells were found to express more lamp-1 and lamp-2 on the cell surface. These results were found to be correlated to the increased expression of sialyl Lex structures with high affinity binding of anti-sialyl Lex antibody on highly metastatic cells. Increased expression of sialyl Lex in the poly-N-acetyllactosamines of the cell surface may contribute to the metastatic behavior of the cells, assuming that this structure can serve as a better ligand for selectins present on endothelial cells and platelets.     

Granulocytic differentiation of HL-60 cells is associated with increase of poly-N-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins

HL-60 cells were induced to differentiate into granulocytic cells by dimethyl sulfoxide, and structures of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins (lamp-1 and lamp-2) were elucidated before and after differentiation. Lamp-1 and lamp-2 were immunoprecipitated from the cells after labeling with radioactive sugars, and glycopeptides were prepared. The structures of glycopeptides obtained after serial lectin-affinity chromatography were elucidated by endo-beta-galactoside and methylation analysis. Glycopeptides bound to tomato lectin-Sepharose were found to be tetraantennary oligosaccharides that contain two or three poly-N-acetyllactosaminyl chains, of which one side chain contains three or more N-acetyllactosaminyl repeats, whereas those bound to Datura stramonium agglutinin-Sepharose were found to be tetraantennary oligosaccharides containing one or two short poly-N-acetyllactosaminyl side chains. Glycopeptides that were not bound to concanavalin A, tomato lectin, or D. stramonium agglutinin were found to be triantennary oligosaccharides with a negligible amount of poly-N-acetyllactosaminyl side chains. Comparison of Asn-linked oligosaccharides from undifferentiated and differentiated HL-60 cells reveals the following features. First, the number of Asn-linked oligosaccharides containing poly-N-acetyllactosaminyl side chains increases dramatically with a concomitant decrease in less complex Asn-linked oligosaccharides after differentiation. Second, the number of poly-N-acetyllactosaminyl side chains per Asn-linked oligosaccharides increases significantly. These increases in poly-N-acetyllactosamine were associated with increased activity of UDP-GlcNAc:beta-D-Gal-beta 1----3-N-acetylglucosaminyltransferase "extension enzyme," a key enzyme in the formation of poly-N-acetyllactosamines. Furthermore, the increased amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 resulted in longer half-lives of lamp-1 and lamp-2 in differentiated HL-60 cells. These results suggest strongly that the differentiation of HL-60 cells into more phagocytic cells is associated with an increase in the complexity of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins, which in turn may play a role in stabilizing lysosomes.     

Cholesterol sulphate sulphohydrolase of human placenta lysosomal membrane

In this paper we report that the activity of cholesterol sulphate sulphohydrolase (CHS-ase) is associated with the lysosomal membranes. The procedure of purification of CHS-ase from human placenta lysosomes was elaborated. The purified enzyme is highly specific to cholesterol sulphate (specific activity 2126.60+/-940.90 nmol min(-1) mg protein(-1)) and acts optimally at pH 3.4. The K(M) value for the hydrolysis of cholesterol sulphate is 3.6+/-0.95 x 10(-5)mol/l. The isoelectric point (pI) has the value 5.7, molecular weight estimated by SDS-PAGE electrophoresis is 38 kDa. The described enzyme may be involved in a regulation of cholesterol and cholesterol sulphate levels in the lysosomal membrane.     

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.     

Comparison of lectin receptor and membrane coat-associated glycoproteins of milk lipid globule membranes.

1. Glycoproteins of bovine (Bos taurus) and human (Homo sapiens) milk lipid globule membranes were characterized by ability to bind lectins after electrophoretic separation. 2. Seven lectin receptor glycoproteins were detected in bovine and five in human milk lipid globule membranes. Bovine and human globule membrane glycoproteins differed in ability to interact with certain lectins. 3. Two major nonionic detergent insoluble glycoproteins were present in bovine and human lipid globule membrane; these constituents had apparent molecular weights of 155,000 and 69,000. Detergent-insoluble polypeptides with similar or identical electrophoretic mobilities were found in milk lipid globule membranes from four other species, rat (Rattus norvegicus), sheep (Ovis aries), pig (Sus scrofa) and goat (Capra hircus). Tryptic peptide mapping revealed these polypeptides to be nonidentical among species.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

Composition and distribution of carbohydrate chains in glycoproteins of human erythrocyte membrane

The M-, N-, and MN-glycoproteins obtained from human erythrocytes by phenol-water extraction were purified by gel filtration and digested with Pronase and trypsin. The products of degradation were fractionated by gel filtration on Sephadex G-25 and DEAE-Sephadex A-50 and the fractions were examined by poly(acrylamide)-gel electrophoresis in the presence of dodecyl sodium sulfate, analyzed for carbohydrate and amino acid contents, and tested for M and N blood-group activity. From the results, it is suggested that the glycoprotein chains are composed of a hydrophobic moiety devoid of carbohydrate chains and a hydrophilic moiety containing carbohydrate chains of different compositions, irregularly distributed along the protein chains and linked to L-asparagine, L-serine, or L-threonine residues. The M and N activity typical for the undegraded glycoproteins, and the “basic” or “precursor-type” N activity, were found in different glycopeptide fractions.     

Study of membrane glycoproteins of human erythrocytes using lectins

A method to study the glycoprotein composition of cell membranes, in particular of human red blood cells, has been developed. It includes the separation of membrane components by the SDS-polyacrylamide gradient slab gel electrophoresis, electroblotting of the phoretograms onto the nitrocellulose sheets and detection of glycoprotein fractions with FITC and peroxidase labeled lectins. PNA detected asialoglycoproteins with O-linked oligosaccharide chains, corresponding to all the PAS-positive bands of the phoretogram. SBA interacted more selectively and revealed only certain PAS-positive bands. Glycoproteins with N-linked carbohydrate chains were PAS-negative and can be identified only by the interaction with WGA, LCL, RCA. Group-specific agglutinins have shown that the ABO antigenic determinants are located in N-linked carbohydrate chains of membrane glycoproteins.     

Regulation of lamp2a levels in the lysosomal membrane

The selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half-life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone-mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone-mediated autophagic pathway.