Structure and function of glycosphingolipids and sphingolipids: recollections and future trends

Abstract

Based on development of various methodologies for isolation and characterization of glycosphingolipids (GSLs), we have identified a number of GSLs with globo-series or lacto-series structure. Many of them are tumor-associated or developmentally regulated antigens. The major question arose, what are their functions in cells and tissues? Various approaches to answer this question were undertaken. While the method is different for each approach, we have continuously studied GSL or glycosyl epitope interaction with functional membrane components, which include tetraspanins, growth factor receptors, integrins, and signal transducer molecules. Often, GSLs were found to interact with other carbohydrates within a specific membrane microdomain termed "glycosynapse", which mediates cell adhesion with concurrent signal transduction. Future trends in GSL and glycosyl epitope research are considered, including stem cell biology and epithelial-mesenchymal transition (EMT) process.

Fig. 1. Stage-specific transition of glycosyl epitopes from globo- to lacto-, and to ganglio-series, during early mouse embryogenesis

Le^x epitope carried by SSEA-1 is not expressed at 4-cell stage, but is maximally expressed at 1.5 – 2 days (8–32 cell; morula stage), and induces Le^x-mediated adhesion (compaction). Le^x disappears after compaction, but is expressed at inner cell mass of blastocyst (3.5 days). Extended globo-series SSEA-3 and -4 (yellow background) are expressed at 0.5 – 1 day, before SSEA-1 appears. Ganglio-series is not expressed until 7 days, when neural crest appears. Data for developmental pattern based on [–87]. Structure for SSEA-1 [87, 88, 216]; structures for SSEA-3 and -4 [53, 54].

Fig. 2. Two approaches used for functional analysis of cell surface glycosyl epitopes and GSLs

Panel A. Analysis of Gal oxidase surface-labeled component by SDS-PAGE column (A-i), or by slab gel electrophoresis with fluorography (A-ii). Samples from NIL cells: A-i, left; A-ii, lanes 1 and 2. Samples from NILpy cells: A-i, right; A-ii, lanes 3 and 4. Note that peak "a" (galactoprotein a) in NIL is lost in NILpy, while peak "c" (galactoprotein b) is greatly enhanced in NILpy. Lane 1, total protein of surface-labeled NIL cells. Lane 2, fluorography of lane 1. Lane 3, surface-labeled NILpy cells, extracted with empigin BB (detergent), purified by RCA (Ricinus communis lectin) and slab gel electrophoresis. Lane 4, fluorography of lane 3. Empigin BB extract contained mainly galactoprotein b (~125 kDa), which is present mainly in NILpy cells. A-iii: Functional domain structure of human plasma fibronectin [217]; for review see [218]. Panel B. Exogenous addition of Gb4 to NIL cells induced growth-inhibited and "contact-oriented" appearance (B-i-b), whereas cells without Gb4 addition grew randomly, with partial overlapping (B-i-a). Synchronized NIL cells were subjected to: determination of cell number increase at G2 phase (B-ii-a), mitotic index (B-ii-b), and thymidine incorporation (B-ii-c). Note that Gb4-added cells did not show cell number increase, and had minimal mitosis and DNA synthesis.

Fig. 2. Two approaches used for functional analysis of cell surface glycosyl epitopes and GSLs

Panel A. Analysis of Gal oxidase surface-labeled component by SDS-PAGE column (A-i), or by slab gel electrophoresis with fluorography (A-ii). Samples from NIL cells: A-i, left; A-ii, lanes 1 and 2. Samples from NILpy cells: A-i, right; A-ii, lanes 3 and 4. Note that peak "a" (galactoprotein a) in NIL is lost in NILpy, while peak "c" (galactoprotein b) is greatly enhanced in NILpy. Lane 1, total protein of surface-labeled NIL cells. Lane 2, fluorography of lane 1. Lane 3, surface-labeled NILpy cells, extracted with empigin BB (detergent), purified by RCA (Ricinus communis lectin) and slab gel electrophoresis. Lane 4, fluorography of lane 3. Empigin BB extract contained mainly galactoprotein b (~125 kDa), which is present mainly in NILpy cells. A-iii: Functional domain structure of human plasma fibronectin [217]; for review see [218]. Panel B. Exogenous addition of Gb4 to NIL cells induced growth-inhibited and "contact-oriented" appearance (B-i-b), whereas cells without Gb4 addition grew randomly, with partial overlapping (B-i-a). Synchronized NIL cells were subjected to: determination of cell number increase at G2 phase (B-ii-a), mitotic index (B-ii-b), and thymidine incorporation (B-ii-c). Note that Gb4-added cells did not show cell number increase, and had minimal mitosis and DNA synthesis.

Fig. 3. α-Gal transferase response (for Gb3 synthesis) upon cell contact in contact-inhibitable BHK and NIL cells, and loss of this response in transformed cells

UDP-Gal: LacCer α-Gal transferase (Gb3 synthase) (○-○) increased significantly with increase of cell population density in both non-transformed BHK and NIL cells (left panels). This response was lost in polyoma virus-transformed BHK and NIL cells (right panels). UDP-Gal: GlcCer β-Gal transferase (LacCer synthase) (●-●) did not increase with increase of cell population density in either BHK or NIL cells, or their transformants. The figure is based on data summarized from [106] and [109].

Fig. 4. Effects of GM3, lyso-GM3, and de-N-acetyl-GM3 on EGFR in A431 cells

When epidermal growth factor (EGF) binds to its receptor (EGFR), cytoplasmic tyrosine kinase is activated [110, 112, 219], possibly through receptor-receptor interaction or some other conformational change of EGFR [220]. EGF-induced receptor tyrosine kinase activity was inhibited by ganglioside GM3 [119], presumably through surrounding EGFR in membrane microdomain. However, lyso-GM3, derived from GM3 by ceramidase, inhibited EGFR tyrosine kinase more strongly than GM3 [120]. De-N-acetyl-GM3, derived from GM3 by de-N-acetylase, strongly promoted receptor kinase [121]. The scheme shown is based on these results.

Fig. 5. Two well-established carbohydrate-to-carbohydrate interactions that mediate homotypic cell adhesion

Top: self-recognition of 3-O-sulfated GlcNAcβ3Fucα-O-Ser/Thr linked to proteoglycan, that mediates species-specific sponge cell adhesion and autoaggregation. Bottom: self-recognition of Galβ4[Fucα3]GlcNAcβ3Gal (Le^x epitope) carried by SSEA-1 glycan, that mediates homotypic adhesion of embryonal stem cells or embryonal carcinoma cells, to induce compaction or autoaggregation. In both cases, presence of Ca²⁺, which promotes interaction of glycan, is essential.

Fig. 6. GSL structures displaying specific CCI

GSL interactions were determined by binding of liposomes containing specific GSL and labeled with ³H-cholesterol to specific GSL coated on polystyrene plate [141, 146, 147]. Strong, weak, or no interaction are indicated by different lines.

Fig. 6. GSL structures displaying specific CCI

GSL interactions were determined by binding of liposomes containing specific GSL and labeled with ³H-cholesterol to specific GSL coated on polystyrene plate [141, 146, 147]. Strong, weak, or no interaction are indicated by different lines.

Fig. 6. GSL structures displaying specific CCI

GSL interactions were determined by binding of liposomes containing specific GSL and labeled with ³H-cholesterol to specific GSL coated on polystyrene plate [141, 146, 147]. Strong, weak, or no interaction are indicated by different lines.

Fig. 7. Multiple Sph-induced mechanisms leading to apoptosis

Apoptotic stimulation causes (i) higher Cer level, followed by release of Sph by ceramidase; and (ii) an unknown mechanism causing translocation of PKCδ and its possible accumulation in mitochondrial membrane. These two events are considered to trigger multiple Sph-induced channels for apoptosis processes as below. a. Sph activates a "cascade of caspases" leading to caspase-3 activation (process 1). b. Activated caspase-3 cleaves PKCδ to produce PKCδ KD (SDK1) (process 2). c. Sph activates SDK1 to phosphorylate 14-3-3 (→14-3-3-P) (process 3). d. 14-3-3-P inhibits 14-3-3 dimer formation (process 4), which in turn inhibits binding of 14-3-3 to pro-apoptotic BAD/BAX, promoting their pro-apoptotic effect (process 5). e. Sph inhibits integrin-dependent survival signal, e.g., Akt (process 6), leading to apoptosis (process 7). f. Sph inhibits BCL-2 gene expression (process 8), thereby inhibiting anti-apoptotic BCL-2/BCL-X (process 9). Processes 5, 7, and 9 cause release of cytochrome c (process 10), which contributes to caspase-3 activation (process 11). g. Activated caspase-3 releases PKCδ KD (SDK1), promoting a "vicious cycle" of Sph-induced apoptosis (process 12). The overall process is based on enhanced Sph level, and translocation of PKCδ. The higher the Sph level, the greater the effect of the cycle through released caspase-3 and released PKCδ KD. Susceptibility of adherent tumor cells to Sph-induced apoptosis is less than that of non-adherent cells, because adherent cells require inhibition of integrin-dependent survival signal by Sph (process 6). Which Sph-induced channel is dominant may vary depending on type of cell. From [182].

Fig. 8. Infertility in women is caused by the presence of antibodies directed to sperm antigens sialyl-i (SA-i) and sialyl-I (SA-I), carried by male-specific CD52

Analysis of gangliosides present in human sperm, and thin-layer chromatography (TLC) immunostaining data with mAb H6-3C4, indicate that the male-specific epitope is sialyl-i, i, or sialyl-I, carried by GPI-anchored CD52 [198, 199] which consists of a short peptide (12 a.a.) with bulky N-linked glycan as shown (sialyl-i, top; i, middle; sialyl-I, bottom). Since H6-3C4 showed stronger reactivity with sialyl 2–6 lactonorhexaosylceramide than with sialyl 2–3 lactonorhexaosylceramide, the sialyl epitope could be a 2–6, 2–3 mixture. Data are explained in the text. From [196].