Ganglioside binding pattern of CD33-related siglecs

Our study deals with the interaction of CD33 related-siglecs-5,-7,-8,-9,-10 with gangliosides GT1b, GQ1b, GD3, GM2, GM3 and GD1a. Siglec-5 bound preferentially to GQ1b, but weakly to GT1b, whereas siglec-10 interacted only with GT1b ganglioside. Siglec-7 and siglec-9 displayed binding to gangliosides GD3, GQ1b and GT1b bearing a disialoside motif, though siglec-7 was more potent; besides, siglec-9 interacted also with GM3. Siglec-8 demonstrated low affinity to the gangliosides tested compared with other siglecs. Despite high structural similarity of CD33 related siglecs, they demonstrated different ganglioside selectivity, in particular to the Neu5Acalpha2-8Neu5Ac motif.     

Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides.

We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGB19, GMR2, GMR7, GGR12, GMR5, and GGR13 reacted strongly with the gangliosides GD3, O-Ac-GD3, GD2, GD1b, GT1b, and GQ1b, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-Ac-GD3, GQ1b, and GT1a, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GD1b, GT1b, and GQ1b on mouse, rat, and human leukemia cells. GD1b was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GT1b nor GQ1b was detected in these cell lines.     

The specificity of monoclonal antibody A2B5 to c-series gangliosides

To examine the specificity of monoclonal antibody A2B5, four A2B5-reactive gangliosides (designated as G-1, G-2, G-3 and G-4) were purified from bonito fish brain. Ganglioside-1, -2, and -3 migrated above GD1b, below GQ1b, and far below GQ1b on thin-layer chromatography. Ganglioside-4 had the slowest chromatographic mobility and migrated below G-3. The structures of these gangliosides were characterized by overlay analysis with glycolipid-specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, G-1, G-2 and G-3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G-4 was shown to have the following structure: NeuAc-NeuAc-NeuAc-Galbeta1-3Gal NAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. The antibody A2B5 reacted with these c-series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato- or ganglio-type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c-series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c-series gangliosides.     

Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

A new thin-layer chromatographic approach for separation of multisialogangliosides: Novel gangliosides fractions in the embryonic chicken brain

A novel thin-layer chromatographic procedure has been developed that permits rapid, high-resolution separation of complex ganglioside mixtures and direct densitometric quantification. A special advantage of the new procedure, performed by two different consecutive runs on high-performance thin-layer chromatography plates, is an excellent separation of multisialogangliosides containing more than three sialic acid residues. Using the new procedure, 10 unidentified fractions were detected in embryonic chick brains. These gangliosides were clearly distinguishable from the known gangliosides, GM1, GD3, GD1a, GD2, GD1b, GT1b, and GQ1b. Eight of these “additional” fractions were also found in the brains of rays. From published data on the cod fish brain, 6 of the novel fractions are suggested to correspond to GT3, GT2, GT1c, GQ1c, GP1c, and GP1b. Four fractions, moving on thin-layer chromatography plates below the suggested GP1c have not been reported previously in any vertebrate. Due to their very slow migration rates they may contain gangliosides with six, seven, or more sialic acid residues. During development of the chicken, the relative amounts of the newly detected fractions decrease in favor of GT1b and GD1a.     

Differential distribution of major gangliosides in rat central nervous system detected by specific monoclonal antibodies

We investigated the localization of major gangliosides in adultrat brain by an immunofluorescence technique with mouse monoclonalantibodies (MAbs). Five MAbs (GMB16, GMR17, GGR12, GMR5 andGMR13) that specifically recognize gangliosides GM1, GD1a, GD1b,GT1b and GQ1b, respectively, were used. We have found that thereis a cell type-specific expression of the ganglioside in therat central nervous system. In cerebellar cortex, GM1 was expressedin myelin and some glial cells. GD1a was detected exclusivelyin the molecular layer. GD1b and GQ1b were present restrictedlyon the granular layer; GD1b was detected on the surface of thegranular cell bodies, whereas GQ1b was present in the cerebellarglomerulus. GT1b was distributed intensely in both the molecularlayer and the granular layer. In cerebral cortex, GM1 was detectedin some glial cells. Dense staining was limited to the whitematter. GD1a was distributed in layers I, II/III and Va, andthe upper part of layer VI, whereas GQ1b was localized in layersIV and Vb, and the lower part of layer VI. GD1b was detectedbeneath layer III. GT1b appeared to be distributed throughoutall layers. In other regions, such as hippocampal formationand spinal cord, the expression of the ganglioside was alsohighly localized to a specific cell type and layer. ganglioside monoclonal antibody rat brain     

Expression of c-series gangliosides in rat hepatocytes and liver tissues

C-series gangliosides in rat hepatocytes and liver tissues were analyzed by thin-layer chromatographic (TLC) immunostaining with the specific monoclonal antibody A2B5. Primary cultures of hepatocytes isolated from adult rats were immunostained positively by A2B5. TLC immunostaining with A2B5 of gangliosides from the cells suggested that rat hepatocytes express c-series gangliosides including GT3, GT1c, GQ1c, and GP1c. Expression of c-series gangliosides in cultured hepatocytes was modulated by growth conditions of cells. The amount of GT3 was increased significantly by epidermal growth factor, while the contents of polysialo species such as GT1c, GQ1c, and GP1c were enhanced by higher cell density in culture. Examination of c-series gangliosides in rat liver tissues showed a unique developmental profile with a shift from GT3-dominant to polysialo species-dominant composition in late embryonic stages. These results suggest that the expression of c-series gangliosides in rat hepatocytes is regulated in a growth- and development-dependent manner.     

Tetrasialoganglioside GQ1b Reactive Monoclonal Antibodies: Their Characterization and Application for Quantification of GQ1b in Some Cell Lines of Neuronal and Adrenal Origin(S)

Seven monoclonal antibodies (MAbs) directed to tetrasialoganglioside (GQ1b) were established, purified GQ1b being used for immunization and hybridoma screening. All of the MAbs reacted strongly with GQ1b, although they also reacted with other gangliosides, with different specificities and reactivities. Some MAbs (1H10, 2C7, and 3F4) reacted with GD3, GT1a, GQ1b, and GP1c. MAb 1H4 showed broad specificity. It reacted with GD3, GD1b, GD2, GT1a, GT1b, GO1b, GQ1c, and GP1c. MAbs 7F5, 4E7, and 4F10 recognized GT1a, GQ1b, and GP1c. MAb 4F10 was more specific for GQ1b than the other MAbs. Using MAb 4F10, we determined, by means of an immunoassay, the quantities of endogenous GQ1b in some neuronal and adrenal cell lines, GOTO (human neuroblastoma), Neuro2a (mouse neuroblastoma), and PC12 (rat pheochromocytoma). PC12 and Neuro2a cells contained at least 5.1 X 10(6) and 3.9 X 10(5) molecules/cell of GQ1b, respectively. In contrast, no GQ1b was detected in GOTO cells, which are known for their specific neuritogenic response to this particular ganglioside when exogenously added.     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.     

Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains

Glycosphingolipids (GSLs) and their sialic acid-containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c-series gangliosides such as GT3 and GQ1c), Chol-1alpha (GT1aalpha and GQ1balpha), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage-specific embryonic antigen-1 (Lewis x) glycolipids, and human natural killer-1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12-E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a-series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage-specific embryonic antigen-1 were expressed in embryonic brains. Human natural killer-1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol-1alpha, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up-regulation of complex a- and b-series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Characterization and Regional Distribution of Individual Gangliosides in Goldfish Central Nervous System

Gangliosides were partially purified from goldfish brain and fractionated by DEAE Fractogel column chromatography. Each fraction was then analyzed by HPTLC and also by HPLC after conversion of the gangliosides to 2,4-dinitrophenylhydrazides. The tetrasialoganglioside GQ1c was found to constitute more than 50% of the total gangliosides. Gangliosides in smaller quantities were also tentatively identified. These included GT1b, GT1c, GT2, GT3, GD1a, and several others. By using this information, the amounts of individual gangliosides in various regions of goldfish central nervous system were compared. Although all areas of brain examined contained similar concentrations of gangliosides, with GQ1c as the predominant component, retina and optic nerve contained significantly lower concentrations of GQ1c, and GM3 was the major component.     

Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.     

Oligosialogangliosides inhibit GM2- and GD3-synthesis in isolated Golgi vesicles from rat liver

The effect of end-product gangliosides (GD1a, GT1b, GQ1b) on the activities of two key enzymes in ganglioside biosynthesis, namely GM2-synthase and GD3-synthase in rat liver Golgi apparatus, has been investigated in detergent-free as well as in detergent-containing assays. In detergent-free intact Golgi vesicles, phosphatidylglycerol was used as a stimulant. This phospholipid was earlier shown to stimulate the activity of GM2-synthase without disrupting the vesicular intactness; it has, however, no effect on GD3-synthase (Yusuf, H.K.M., Pohlentz, G., Schwarzmann, G. & Sandhoff, K. (1983) Eur. J. Biochem. 134, 47-54). In the presence of this stimulant, all higher gangliosides inhibited the activity of GM2-synthase, the inhibition being more profound with increasing negative charge of the inhibiting gangliosides. These inhibitions are unspecific, but they do not exclude an end-product regulation of ganglioside biosynthesis. In detergent-solubilized Golgi membranes, on the other hand, the inhibition pattern was completely different. Here, ganglioside GD1a was the strongest inhibitor of GM2-synthase, followed by GM1 and GM2, but GT1b also inhibited this enzyme appreciably, in fact more strongly than GM1 or GM2. On the other hand, GQ1b had no effect at all. Conversely, GD3-synthase activity was most strongly inhibited by GQ1b, followed by GT1b, but GD1a also inhibited this enzyme almost as strongly as GT1b. These latter findings indicate that feed-back control of the a- and the b-series pathways of ganglioside biosynthesis is probably not specific, but the pathways appear to be inhibited more preferably by their respective end-products than by any other gangliosides of the same of the other series.     

Genetic polymorphism of rat liver gangliosides

Liver ganglioside patterns of eight rat strains were classified according to two phenotypes: SHR type, characterized by predominance of b-series gangliosides (GD1b, GT1b, GQ1b), and DA type, characterized by predominance of a-series gangliosides (GM1, GD1a). Comparison of ganglioside pattern expressed in the liver of F1 hybrids and backcross F2 hybrids indicated that SHR type is controlled by a single autosomal-dominant gene which probably determines the expression of sialytransferase 2 activity for synthesis of GD3 from GM3.     

Tissue-specific expression of c-series gangliosides in the extraneural system

c-Series gangliosides in extraneural tissues from young and adult rats were examined using thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody A2B5. The composition of c-series gangliosides significantly differed among tissues. In adult rats, while liver tissue contained GT1c, GQ1c, and GP1c, renal tissue had GT3 as the major c-series ganglioside with GT2 in a lesser amount. Pancreatic tissue expressed c-series gangliosides that consisted of GT3, GT2, GQ1c, and GP1c. In other tissues including adrenal, thyroid, and eye lens, GT3 constituted the main c-series ganglioside species. While total ganglioside contents of extraneural tissues were much lower than that of brain tissue, the proportions of c-series gangliosides to total gangliosides were higher in many extraneural tissues. Interestingly, eye lens had the highest GT3 content among rat tissues examined. The compositions and concentrations of c-series gangliosides in liver and kidney significantly differed between 5-day-old and 7-week-old rats, suggesting the development-dependent expression of c-series gangliosides in these tissues. These results suggest that the expression of c-series gangliosides in extraneural tissues is regulated in a tissue-specific manner.     

Antibodies against gangliosides and ganglioside complexes in Guillain–Barré syndrome: New aspects of research

Guillain–Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.