Recycling MHC class I molecules and endosomal peptide loading.

MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH(4)Cl-sensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.

Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective purification of plasma membranes.

Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.

Electromigration for separations of protein complexes.

This paper describes electromigration of complexes, consisting of two or more proteins and non-covalently associated peptides. Relatively small complexes (Mr < 1000000) can be resolved in sieving matrices. Large complexes are separated in free liquid systems. Examples of separation are given using native gels, denaturing gels and special formats thereof: blue native PAGE and gels incorporating a transversal temperature gradient. Both preparative and analytical applications are discussed as well as separations leading to mechanistic models of protein interaction. Carrier-free electrophoresis is represented by capillary zone electrophoresis, free-flow electrophoresis and density gradient electrophoresis. Emphasis is put on the free liquid separation of clathrin-coated vesicles and proteasomes.

High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions.

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.

High performance density gradient electrophoresis of subcellular organelles, protein complexes and proteins.

A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.

Removal and degradation of the free MHC class II beta chain in the endoplasmic reticulum requires proteasomes and is accelerated by BFA.

We have studied the degradation of the free major histocompatibility complex (MHC) class II beta subunit in the ER. Domain swapping experiments demonstrate that both the intra- and extracellular domain determine the rate of degradation. Recently, it has been shown that some ER-retained proteins are exported from the ER by the translocon followed by deglycosylation and degradation in the cytosol by proteasomes. Degradation of the beta chain follows a different route. The proteasome is involved but inhibition of the proteasome by lactacystin does not result in deglycosylation and export to the cytosol. Instead, the beta chain is retained in the ER implying that extraction of the beta chain from the ER membrane requires proteasome activity. Surprisingly, brefeldin A accelerates the degradation of the beta chain by the proteasome. This suggests that various processes outside the ER are involved in ER-degradation. The ER is the site from where misfolded class II beta chains enter a proteasome-dependent degradation pathway.

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.

Density gradient isoelectric focusing of proteins in artificial pH gradients made up of binary mixtures of amphoteric buffers.

A density gradient electrophoresis apparatus made of Perspex (7 cm, O 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bier et al. (Electrophoresis 1993, 14, 1011-1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK(1,1)-1-2) and the pK(2,II)+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and gamma-amino-n-butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate-zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochrome c, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate-zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycoforms of transferrin were separated and enriched on a pH 5.2-6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill-defined Ampholines, the cost of these well-defined amphoters is low.

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading.

BACKGROUND: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide 'editor', favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive. RESULTS: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM. CONCLUSIONS: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.

High-resolution density gradient electrophoresis of proteins and subcellular organelles.

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.

Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface.

Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.

HLA-DM and MHC class II molecules co-distribute with peptidase-containing lysosomal subcompartments.

MHC class II molecules associate with peptides in the endocytic pathway. Different endosomal locations for peptide loading of class II molecules, varying from early endosomes (EE) to lysosomes, have been assigned on the basis of subcellular fractionation experiments. We have determined the intracellular location of HLA-DM, a molecule that supports peptide loading of class II molecules, by separating vesicles from the melanoma cell line Mel JuSo on the basis of buoying density and surface charge. In both fractionations, HLA-DM co-fractionated with a lysosomal compartment containing beta-hexosaminidase (beta-hex) activity and not with endosomes. Further analysis showed that HLA-DM mainly co-fractionated with a sub-lysosomal structure characterized by a relative low density and containing both pro- and mature cathepsin D and MHC class II molecules. Fluid phase markers first enter this compartment before entering high-density lysosomes that contain exclusively mature cathepsin D, some HLA-DM and no detectable MC class II molecules. Finally we determined the intracellular location of neutral and acidic peptidases. Whereas neutral peptidase activity was detected in the endoplasmic reticulum and/or plasma membrane fractions, acidic peptidase activity exclusively migrated at the position of HLA-DM containing lysosomal vesicles. Our results show that class II molecules co-migrate with HLA-DM, pro- and mature cathepsin D, beta-hex and acidic peptidase activity. HLA-DM, cathepsin d and class II molecules were not observed at the position of EE. Our data suggest that HLA-DM-mediated peptide loading of class II molecules occurs in a lysosomal subcompartment.

High resolution density gradient electrophoresis of cellular organelles.

A density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 x 2.2 cm) containing a 0-5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O-ring. The top circular platinum anode allows for upward electrophoresis (80-100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle-enclosed enzymes.

Distinct populations of high-M(r) mucins secreted by different human salivary glands discriminated by density-gradient electrophoresis.

High-M(r) mucins [mucin glycoprotein 1 (MG1)] isolated from human saliva from the individual salivary glands were chemically characterized. The carbohydrate content of MG1 derived from palatal (PAL), submandibular (SM) and sublingual (SL) saliva was typical of mucins but showed heterogeneity, especially in the amount of sialic acid and sulphated sugar residues. The physicochemical properties of native MG1s make conventional SDS/PAGE and ion-exchange chromatography unsuitable for investigating differences between individual samples. Recently a density-gradient electrophoresis (DGE) device has been developed, primarily for separation based on the charge of entire cells or cell organelles [Tulp, Verwoerd and Pieters (1993) Electrophoresis 14, 1295-1301]. We have used this apparatus to study the high-M(r) salivary mucins. Using DGE, the MG1s of individual glands were seen to have clearly distinct electrophoretic mobilities, as monitored by ELISA using MG1-specific monoclonal antibodies. Even within a particular MG1 preparation, subpopulations could be distinguished. DGE analysis of a chemically and enzymically modified MG1 series, followed by ELISA and dot-blot detection using specific monoclonal antibodies, lectins and high-iron diamine staining, suggests that the high electrophoretic mobility of PAL-MG1 is mainly the result of a high sulphate content, whereas the SL subpopulations differ mainly in binding type and amount of sialic acid. SM-MG1 most resembles the low-mobility subpopulation of SL-MG1, except that it has a lower sulphate content. In conclusion, DGE appears to be a powerful method for analysis of native mucin; it has been used to demonstrate that MG1s from the various salivary glands are biochemically much more diverse than was previously assumed.

Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments.

The HLA-DM genes encode an unconventional HLA (human leukocyte antigen) class II molecule that is required for appropriate binding of peptide to classical HLA class II products. In the absence of DM, other class II molecules are unstable upon electrophoresis in sodium dodecyl sulfate and are largely associated with a nested set of peptides derived from the invariant chain called CLIP, for class II-associated invariant chain peptides. DMA and DMB associated and accumulated in multilaminar, intracellular compartments with classical class II molecules, but were found infrequently, if at all, at the cell surface. Thus, DM may facilitate peptide binding to class II molecules within these intracellular compartments.

Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Isolation and characterization of the intracellular MHC class II compartment.

An intracellular compartment has been isolated to which MHC class II molecules are transported on their way to the plasma membrane. They arrive with an associated invariant chain which is then proteolytically processed while MHC class II molecules acquire antigenic peptide. These loaded class II molecules then leave the compartment devoid of invariant chain and bound for the plasma membrane. This compartment represents a new stage in the endocytic/lysosomal pathway.

Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles.

A DGE apparatus, made of Perspex, consisting of a separation column (5 x 2.2 cm) and containing a 0-4% linear Ficoll density gradient, was constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3-1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cation-permeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30-60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of a1-antitrypsin were separated as well as isoenzymes of beta-hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf-receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 10(7) cells (of 100 microns3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE):lysosomes (L):Golgi (G):early endosomes (EE) = 1:0.94:0.77:0.55 and under slightly different conditions LE:L:G:endoplasmatic reticulum (ER):plasma membrane (PM) = 1:0.87:0.64:0.58:0.49.