CELL SURFACE PROTEINS OF CULTURED BRAIN CELLS AND THEIR RECOGNITION BY ANTI-CEREBELLUM (ANTI-NS-4) ANTISERUM

Abstract— Surface proteins of cultured young postnatal mouse cerebella and embryonic mouse cerebral hemispheres were identified by Iactoperoxidase-catalysed radioiodination and by their interaction with an anti-mouse cerebellum antiserum (anti-NS-4 serum) which recognizes surface components on brain cells. Several (8 10) iodinated polypeptides are recognized by radioautography after polyacrylamide gel electrophoresis. Their surface location was confirmed by their sensitivity to mild trypsin treatment on intact cells. Iodinated polypeptides from cells of non-nervous tissues showed a different gel pattern. Immuno-precipitates of solubilizcd surface-iodinated cerebellar cells with anti-NS-4 serum contained two prominent labeled proteins with apparent molecular weights of 200 × 10³ and 145 × 10³. These proteins were also biosynthetically labeled with [³H]leucine. The 145 × 10³ molecular weight component was also found in immunoprecipitates prepared from embryonic cerebral cells, but the 200 × 10³ molecular weight component was replaced by a broad peak with an apparent molecular weight of around 250 × 10³.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane

Mouse leukemia L-1210 cells were iodinated with ¹²⁵I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of ¹²⁵I over that of the cell homogenate and a 20-fold increase in the specific activities of 5′-nucleotidase and alkaline phosphate; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide ($\text{M}{}_{\mathrm{<mtext>m</mtext>}}$ 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel sytem for the analysis of membrane proteins is emphasized.     

HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.     

Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture

Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from $\text{> 200 000}\text{to}\text{12 500}$. Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95 000 and 78 000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[¹²⁵I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin.     

Central nervous system antigen P84 can serve as a substrate for neurite outgrowth

Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes. Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule. Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa. When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth. Glial cell attachment was also observed. Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate. This antigen is found on the surfaces of neurons in cultures of cerebellar cells. It is also found on a subclass of unidentified flat cells. P84 is not found on oligodendrocytes or GFAP-positive astrocytes. As early as E9, P84 could be detected in the floor plate region of the spinal cord. This pattern persists throughout embryonic development. Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane.

Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide (Mr 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel system for the analysis of membrane proteins is emphasized.     

Identification of exposed surface glycoproteins of four human bladder carcinoma cell lines

Three cell surface protein-specific methods were used to radiolabel the major glycoproteins of four human bladder carcinoma cell lines: The well-differentiated lines RT112 and TR4 and more anaplastic lines T24 and EJ. Five acidic glycoproteins iodinated in all lines by the lactoperoxidase/125I method were designated CP-175/5.8-6.0 (apparent molecular weight X 10(-3)/pl of iodoprotein), GP-155/5.0-5.3, GP-145/4.9-5.2, GP-130/4.8-5.5 and GP-110/4.9-5.3. Another iodinated glycoprotein, GP-200/5.5-6.0, was prominently labelled in RT112 and RT4 but was not detected in T24 or EJ. GP-200 as well as GP-175, GP-155 and GP-145 were not detected by the galactose oxidase/NaB(3H)4 method and were poorly labelled by the neuraminidase-galactose oxidase/NaB(3H)4 and NaIO4/NaB(3H)4 labelling methods. The major sialogalactoproteins identified in the four lines by the neuraminidase-galactose oxidase/NaB(3H)4 and NaIO4/NaB(3H)4 methods were GP-130, and a duplet of GP-90 and GP-80 which were poorly iodinated by lactoperoxidase/125I. The galactose oxidase/NaB(3H)4 reaction was increased by between 4- and 10-fold and many additional glycoproteins were labelled after neuraminidase treatment, indicating that the cell surface galactose and N-acetylgalactosamine residues of glycoproteins are highly sialylated. In cell lines RT112 and RT4 there was prominent labelling of very high molecular weight sialogalactoconjugates that was not present in extracts of T24 and EJ.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.     

Bacteriophage T4 tail assembly: structural proteins and their genetic identification

We have identified the structural proteins of phage T4 precursor tails. Complete tails, labeled with ¹⁴C-labeled amino acids, were isolated from cells infected with mutants blocked in head assembly. The proteins were characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis and subsequent autoradiography. The complete tails are made up of at least fifteen different species of phage proteins.To identify the genes specifying these proteins we prepared ¹⁴C-labeled amino acid lysates made with amber mutants defective in each of the twenty-one genes involved in tail assembly. Comparison of the gel pattern of the amber mutant lysates with wild type lysates enabled us to identify the following gene products, with molecular weights in parentheses: P6 (85,000); P7 (140,000); P8 (46,000); P9 (34,000); P10 (88,000); P11 (26,000); P12 (55,000); P15 (35,000); P18 (80,000); P19 (21,000); P29 (77,000). These eleven species are all structural proteins of the tail. The genetically unidentified tail proteins have molecular weights of 42,000, 41,000, 40,000 and 35,000. They are likely to be the products of known phage genes which were not resolved in the crowded middle region of the whole lysate gel patterns. The major tail proteins are all synthesized during the late part of the phage growth cycle.The mobilities of the proteins derived from tails did not differ from the mobilities of the proteins when derived from the unassembled pools of subunits accumulating in mutant infected cells, or when derived from complete phage particles.The genes for at least seven of the structural proteins are contiguous on the genetic map. Genes for proteins needed in many copies seem to be clustered separ- ately from genes whose products are needed in only a few copies. Consideration of protein sizes and published mapping data on phage T4 also suggest that the phage structural proteins are, on the average, much larger than the non-structural proteins.The requirement that at least fifteen different species of proteins must come together in forming a phage tail emphasizes the complexity of this morphogenetic process.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside-out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins lableled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic ³²P. We conclude that the phagolysosome is probably oriented in an “inside-out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Cytokeratin expression during mouse embryonic and early postnatal mammary gland development

Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6⁺ and K8⁺/K14⁺ putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.     

Glycoprotein metabolism in developing mouse brain

—Incorporation of [¹⁴C]fucose or [¹⁴C]glucosamine into the glycoproteins of developing mouse brain was studied using polyacrylamide gel electrophoresis. Between 1 and 10 days after birth two fractions of soluble glycoproteins were extensively labelled, but by 15 days after birth incorporation into these fractions was no longer prominent. These glycoproteins have apparent molecular weights in the range of 150,000-250,000, as estimated by the electrophoretic procedure. The more rapidly migrating fraction has a half-life of about 1 week whereas the other is far more stable.     

Overactivation of Notch1 signaling induces ectopic hair cells in the mouse inner ear in an age-dependent manner

Background

During mouse inner ear development, Notch1 signaling first specifies sensory progenitors, and subsequently controls progenitors to further differentiate into either hair cells (HCs) or supporting cells (SCs). Overactivation of NICD (Notch1 intracellular domain) at early embryonic stages leads to ectopic HC formation. However, it remains unclear whether such an effect can be elicited at later embryonic or postnatal stages, which has important implications in mouse HC regeneration by reactivation of Notch1 signaling.

Methodology/Principal Findings

We performed comprehensive in vivo inducible overactivation of NICD at various developmental stages. In CAG^CreER+; Rosa26-NICD^loxp/+ mice, tamoxifen treatment at embryonic day 10.5 (E10.5) generated ectopic HCs in the non-sensory regions in both utricle and cochlea, whereas ectopic HCs only appeared in the utricle when tamoxifen was given at E13. When tamoxifen was injected at postnatal day 0 (P0) and P1, no ectopic HCs were observed in either utricle or cochlea. Interestingly, Notch1 signaling induced new HCs in a non-cell-autonomous manner, because the new HCs did not express NICD. Adjacent to the new HCs were cells expressing the SC marker Sox10 (either NICD+ or NICD-negative).

Conclusions/Significance

Our data demonstrate that the developmental stage determines responsiveness of embryonic otic precursors and neonatal non-sensory epithelial cells to NICD overactivation, and that Notch 1 signaling in the wild type, postnatal inner ear is not sufficient for generating new HCs. Thus, our genetic mouse model is suitable to test additional pathways that could synergistically interact with Notch1 pathway to produce HCs at postnatal ages.     

Adhesive specificity of developing cerebellar cells on lectin substrata

Ten lectins, each with a different carbohydrate-binding specificity, have been coupled to tissue culture substrata with carbodiimide [1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate] and assayed for their efficacy as substrates for the carbohydrate-specific adhesion of cells dissociated from mouse cerebellum at embryonic Day 13 and postnatal Days 0 and 7. On surfaces treated with concanavalin A, succinyl-concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin, both embryonic and early postnatal cerebellar cells formed a monolayer. On surfaces coupled with Ricinus communis_I agglutinin (120,000 daltons) both embryonic and postnatal cells formed cellular aggregates with extensive fiber outgrowth. On surfaces treated with peanut agglutinin, Dolichos bifloris agglutinin, Wistaria floribunda agglutinin, soybean agglutinin, or Ulex europaeus_I agglutinin, embryonic cerebellar cells formed cellular aggregates with a cell viability of 25–35% and little or no fiber outgrowth. Postnatal cerebellar cells, in contrast, formed cellular aggregates with a cell viability of 60–70% and extensive fiber outgrowth. On surfaces treated with Ulex europaeus_I agglutinin, cells from postnatal Day 7 formed limited areas of monolayer in addition to cellular aggregates. After 12 hr in vitro the specific attachment of cerebellar cells to lectin-derivatized substrata was inhibited 60–80% by the inclusion of free hapten carbohydrate (50–100 mM) in the growth medium. The addition of soluble concanavalin A or Ricinus communis_I agglutinin (100 μg/ml) was toxic. These studies suggest the presence of glycoconjugate-binding sites for concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin which promote cerebellar cellular adhesion.     

Schwann Cell Surface Proteins and Glycoproteins

Abstract: To identify surface sialoglycoproteins of rat Schwann cells and to compare molecular weights of these sialoglycoproteins with those present in rat peripheral nervous system myelin, we prepared Schwann cells from sciatic nerves of 1–3-day-old rats and cultured them in monolayer. Surface sialoglycoproteins of the cultured cells were tritium-labeled by the periodateborohydride procedure and compared with sialoglycoproteins of adult rat peripheral nervous system myelin by fluorography following polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate. Three radioactive bands with apparent molecular weights of 114,000–132,000, 105,000–115,000, and 44,000–56,000 were observed in both the Schwann cell and myelin preparations. Bands of similar apparent molecular weights were noted in Schwann cells metabolically radiolabeled with d -[1,6-³H]glucosamine. A band co-migrating with myelin P₀ glycoprotein was the most intensely radiolabeled of all peptides in periodate-B³H₄^?treated myelin, but was present in only trace amounts in periodate-B³H₄^? or d -[1,6-³H]glucosamine radiolabeled Schwann cells. Many presumably non-myelin glycoproteins were identified in the cultured Schwann cells by the periodate-borohydride procedure and by incubation of the cells with d -[1,6-³H]glucosamine. An immunoprecipitation technique was used to detect radiolabeled peptides in a nonionic detergent extract of freshly prepared, surface-radioiodinated Schwann cells that were bound by a rabbit anti-Schwann cell serum preabsorbed with rat fibroblasts. Many radioactive peptides were detected in the immunoprecipitate, but the two most intensely radiolabeled had apparent molecular weights of 105,000–115,000 and 95,000–106,000. This study has identified a number of glycoproteins synthesized by cultured rat Schwann cells which resemble in apparent molecular weight the glycoproteins expressed in rat peripheral nervous system myelin and has defined Schwann cell surface proteins recognized by a specific anti-rat Schwann cell antiserum.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Serological characterization of a nervous system/sperm antigen (NS-52): demonstration of its presence on murine preimplantation embryos.

Anti-NS-5 antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice could be shown to recognize two cell surface antigens on cerebellar cells, NS-5₁ and NS-5₂, the latter antigen being shared with mouse and rat but not rabbit sperm. An antigen operationally identical to NS-5₂ was detected using indirect immunofluorescence staining on mouse preimplantation stages of development. While the unfertilized ova did not express detectable antigen on the cell surface, the fertilized egg expressed antigen shortly before the first cleavage division. From that stage onward, the anti-NS-5 antiserum stained the blastomeres of all stages, including the trophoblast cells and inner cell mass cells of the blastocyst. No difference in staining activity was observed for preimplantation embryos of various mouse strains analyzed: C57BL/6J, BALB/c, 129/J, C3H/DiSn, CKB × BALB.K, C3H.SW/Sn, and Swiss Webster mice. The staining activity was removed when the antiserum was preabsorbed with cerebellum or sperm from any of these mouse strains or with cerebellum and sperm of rats. Lymphocytes, thymocytes, liver, kidney, and skeletal muscle from early postnatal and adult mice and heart from early postnatal mice did not absorb the staining activity and neither did rabbit sperm nor cerebellum.     

A LewisX Glycoprotein Screen Identifies the Low Density Lipoprotein Receptor-related Protein 1 (LRP1) as a Modulator of Oligodendrogenesis in Mice

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.     

Accessibility of plasma membrane sialoglycoconjugates of novikoff tumor cells to exogenous neuraminidase and proteases

Plasma membrane glycoconjugates of Novikoff tumor cells were radioactively labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄ Submission of the radioactively labeled glycoconjugates to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by fluorography revealed the presence of at least ten major glycoproteins and a glycolipid fraction. The glycolipid fraction contained 34% of the cell-surface radioactive label. Pretreatment of cells with neuraminidase from Vibrio cholerae reduced radioactive labeling of the glycoproteins by 71% and that of the glycolipids by 39%. Sequential treatment of cells with papain and neuraminidase further reduced radioactive labeling of the glycolipid fraction, indicating that resistance of this fraction to the hydrolytic action of neuraminidase was determined, at least in part, by steric factors. Incubation of cells with papain resulted in extensive degradation of most of the radioactively labeled glycoproteins with the exception of a subset of glycoproteins having apparent molecular weights of $\text{48 000 ± 5000}$. Trypsin was more selective, degrading three glycoproteins having apparent molecular weights of 200 000, 140 000 and 37 000.