Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.     

Structure and biosynthesis of prokaryotic glycoproteins

Glycoproteins as components of cell surfaces are not restricted to eukaryotes. The prokaryotic glycoprotein studied in greatest detail so far is the cell surface glycoprotein of the archaebacterium Halobacterium halobium. This bacterial glycoprotein contains 3 different types of glycoconjugates, and each type of glycoconjugate involves a different carbohydrate-protein linkage unit: 1) One glycosaminoglycan chain, constructed from a repeating sulfated pentasaccharide block, is linked to one protein molecule via the novel N-glycosyl linkage unit asparaginyl-N-acetylgalactosamine. 2) Ten sulfated oligosaccharides that contain glucose, glucuronic acid and iduronic acid are bound to the protein via the hitherto unknown N-glycosyl linkage unit asparaginylglucose. 3) About 15 disaccharides, glucosylgalactose, are O-glycosyl-linked to a cluster of threonine residues close to the C-terminus of the core protein. The overall structure of the cell surface glycoprotein of halobacteria is thus reminiscent of animal proteoglycans and a functional role of the glycosaminoglycan chain in maintaining the rod shape of halobacteria is discussed. Biosynthesis of the two N-glycosyl linkage units involves dolichol monophosphate and dolicholdiphosphate-linked saccharide precursors. Sulfation and epimerization of the glycoconjugates occur at the lipid-linked level and the mature saccharides are transferred to the protein core on the cell surface. The sulfated oligosaccharides that finally become bound to asparagine via glucose are transiently methylated at their lipid-linked stage and this transient chemical modification seems to be required for the biosynthesis of the corresponding N-glycosyl bond.     

Identification and function of transmembrane glycoproteins--the red cell model

Transmembrane glycoproteins in the red cell membrane traverse the plasma membrane, have their carbohydrate moieties on the extracellular surface, are sialyated (except for band 3) and are tethered to the membrane cytoskeleton proteins on the cytoplasmic surface. This linkage between the transmembrane proteins and the skeletal membrane proteins provides a two-way communication between the extracellular surface and the interior of the red cell; i.e., a transmembrane effect can be initiated from either side. These interactions are discussed in this review, including the example of sickle cell anemia in which the membrane bound hemoglobin may exert a transmembrane effect to change the conformation or distribution of transmembrane glycoproteins and and hence the extracellular surface receptors. This, in turn, may explain why sickle cells adhere to endothelium in vitro. Although the RBC transmembrane sialoglycoproteins may function in communication, regulation of cell shape, and adhesion, uncertainties exist regarding many of their functions. To study these sialoglycoproteins, we have developed a double staining technique (Dzandu et al., 1984) that differentially stains human RBC membrane sialoglycoproteins and asialoproteins in SDS-polyacrylamide gels. This should aid in elucidating the conformational structure and function of transmembrane glycoproteins.     

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.     

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium

The archaeon Halobacterium halobium expresses a cell surface glycoprotein (CSG) with a repeating pentasaccharide unit N- glycosidically linked via N-acetylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type). This aspar-agine of the linkage unit is located within the N-terminal sequence Ala-Asn-Ala-Ser- , in accordance with the tripeptide consensus sequence Asn-Xaa-Ser/Thr typical for nearly every N-glycosylation site known so far, which are of the GlcNAc(1-N)-Asn linkage type. By a gene replacement method csg mutants were created which replace the serine residue of the consensus sequence by valine, leucine, and asparagine. Unexpectedly, this elimination of the consensus sequence did not prevent N-glycosylation. All respective mutant cell surface glycoproteins were N-glycosylated at Asn-2 with the same N-glycan chain as the wild type CSG. Asn-479 is N- glyco-sylated via a Glc(1-N)Asn linkage type in the wild type CSG. Replacement of Ser-481 in the sequence Asn-Ser-Ser for valine prevented glycosylation of Asn-479. From these results we postulate the existence of two different N-glycosyltransferases in H.halobium, one of which does not use the typical consensus sequence Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.      

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60, whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5). The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins.     

Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line.

Two glycoproteins, GP-1 and GP-2, have been isolated from an extracellular membrane synthesized in cell culture by an embryonal carcinoma-derived cell line. The amino acid and carbohydrate compositions have been determined. Both proteins are rich in half-cystine residues and contain approximately 12-15% carbohydrate. Antibodies have been obtained against one of the glycoproteins, GP-2, in rabbits. The antibody reacts with basement membranes from adult mouse and human kidney glomeruli and tubules, and all basement membranes tested from mouse embryonic tissues. The molecular properties of GP-2 are superficially similar to LETS protein; however, immunological and other criteria show that they are distinct proteins. The presence of LETS protein and GP-2 in basement membranes suggests that there are subtle interactions which are important in adhesion of epithelial cells to basement membranes.     

A soluble form of Wnt-1 protein with mitogenic activity on mammary epithelial cells.

The proto-oncogene Wnt-1 plays an essential role in fetal brain development and causes hyperplasia and tumorigenesis when activated ectopically in the mouse mammary gland. When expressed in certain mammary epithelial cell lines, the gene causes morphological transformation and excess cell proliferation at confluence. Like other members of the mammalian Wnt family, Wnt-1 encodes secretory glycoproteins which have been detected in association with the extracellular matrix or cell surface but which have not previously been found in a soluble or biologically active form. We show here that conditioned medium harvested from a mammary cell line expressing Wnt-1 contains soluble Wnt-1 protein and induces mitogenesis and transformation of mammary target cells. By immunodepletion of medium containing epitope-tagged Wnt-1, we show that at least 60% of this activity is specifically dependent on Wnt-1 protein. These results provide the first demonstration that a mammalian Wnt protein can act as a diffusible extracellular signaling factor.     

The C-Terminal Repeating Units of CsgB Direct Bacterial Functional Amyloid Nucleation

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β-strand-loop-β-strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the first, second, or third repeating units were able to convert CsgA into fibers. However, mutants missing either the fourth or fifth repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2, and 4 self-assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.     

Sequence of the halobacterial glycosaminoglycan

The cell-surface glycoprotein of halobacterium contains a sulfated repeating unit saccharide chain, similar to the mammalian glycosaminoglycans. The composition of a presumptive repeating pentasaccharide unit of this glycosaminoglycan is 1 GlcNAc, 1 GalNAc, 1 Gal, 1 GalA (where GalA represents galacturonic acid), 1 3-O-methyl-GalA, and 2 SO42-. Linkage to protein of this glycoconjugate involves the hitherto unique unit Asn-GalNAc, with the N-linked asparagine residue being the second NH2-terminal amino acid and part of the common N-linked glycosyl acceptor sequence Asn-X-Thr(Ser). Transfer of the completed, sulfated glycosaminoglycan from its lipid precursor to the protein occurs at the cell surface, and the presence of this sulfated saccharide chain in the cell-surface glycoprotein seems to be required to maintain the structural integrity of the rod-shaped halobacteria. In this paper, we report the complete saccharide structure of this N-linked glycosaminoglycan. This structure is deduced from chemical analyses of fragments that were isolated after hydrazinolysis and subsequent nitrous acid deamination or after mild acidic hydrolysis of purified Pronase-derived glycosaminoglycan-peptides. The halobacterial glycosaminoglycan consists, on the average, of 10 repeating pentasaccharide units of the following structure. (formula: see text) The reducing end N-acetylgalactosamine residue is linked directly to the asparagine, without a special saccharide linker region.     

The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity

Pathfinding of axons in the developing nervous system is thought to be mediated by glycoproteins expressed on the surface of embryonic axons and growth cones. One molecule suggested to play a role in axonal growth is TAG-1, a 135 kd glycoprotein expressed transiently on the surface of subsets of neurons in the developing mammalian nervous system. We isolated a full-length cDNA clone encoding rat TAG-1. TAG-1 has six immunoglobulin-like domains and four fibronectin type III-like repeats and is structurally similar to other immunoglobulin-like proteins expressed on developing axons. Neurons maintained in vitro on a substrate of TAG-1 extend long neurites, suggesting that this protein plays a role in the initial growth and guidance of axons in vivo. TAG-1 is anchored to the neuronal membrane via a glycosyl phosphatidylinositol linkage and is also released from neurons, suggesting that TAG-1 also functions as a substrate adhesion molecule when released into the extracellular environment.     

Laminin and fibronectin promote the haptotactic migration of B16 mouse melanoma cells in vitro

The migration of tumor cells through basement membranes and extracellular matrices is an integral component of tumor invasion and metastasis. Laminin and fibronectin are two basement membrane- and extracellular matrix-associated noncollagenous glycoproteins that have been shown to promote both cell adhesion and motility. Purified preparations of laminin and fibronectin stimulated the directed migration of B16 murine metastatic melanoma cells in vitro as assessed in modified Boyden chambers. The stimulation of migration occurred over a concentration range of 1-100 micrograms/ml of laminin or fibronectin, with a peak response occurring between 12.5 and 25 micrograms/ml. The maximal response of these cells was 80-120-fold higher than control migration. Affinity-purified antibody preparations specifically abrogated the migration of these cells in response to the respective proteins. Tumor cells in suspension were preincubated in physiologic levels of plasma fibronectin prior to assay to partially mimic what occurs when a metastasizing cell is in the blood stream. This preincubation with plasma fibronectin had no effect on the subsequent migration of cells in response to either laminin or fibronectin. Furthermore, experiments using filters precoated with fibronectin or laminin indicated that these cells could migrate by haptotaxis to these two proteins. We conclude that tumor cell migration in response to such noncollagenous adhesive glycoproteins could be an important aspect in the invasion and metastasis of certain malignant cell types.     

Bridging structure with function: structural, regulatory, and developmental role of laminins

The basement membrane is a highly intricate and organized portion of the extracellular matrix that interfaces with a variety of cell types including epithelial, endothelial, muscle, nerve, and fat cells. The laminin family of glycoproteins is a major constituent of the basement membrane. The 16 known laminin isoforms are formed from combinations of alpha, beta, and gamma chains, with each chain containing specific domains capable of interacting with cellular receptors such as integrins and other extracellular ligands. In addition to its role in the assembly and architectural integrity of the basement membrane, laminins interact with cells to influence proliferation, differentiation, adhesion, and migration, processes activated in normal and pathologic states. In vitro these functions are regulated by the post-translational modifications of the individual laminin chains. In vivo laminin knockout mouse studies have been particularly instructive in defining the function of specific laminins in mammalian development and have also highlighted its role as a key component of the basement membrane. In this review, we will define how laminin structure complements function and explore its role in both normal and pathologic processes.     

Identification and characterization of a novel antigen complex on mouse mammary tumor cells using a monoclonal antibody against platelet glycoprotein Ic

The rat monoclonal antibody GoH3 identifies a complex of glycoproteins Ic and IIa on human and mouse platelets. The GoH3 epitope is located on glycoprotein Ic. A novel glycoprotein complex is identified by GoH3 on the surface membranes of mouse mammary epithelial tumor cells. This antigen complex is composed of glycoprotein Ic noncovalently associated with a monomor or a disulfide-linked multimer of a high molecular weight glycoprotein (Ic-binding protein (IcBP]. Glycoprotein Ic is synthesized as a large precursor with asparagine N-linked high mannose oligosaccharides. Processing of this precursor involves a proteolytic cleavage of the large polypeptides into two smaller disulfide-linked polypeptide chains, Ic alpha (heavy) and Ic beta (light), and conversion of the majority of the high mannose oligosaccharides into complex-type glycans. Likewise, glycoprotein IcBP is initially glycosylated with high mannose asparagine N-linked oligosaccharides which are processed to complex units in the mature form. Association of glycoprotein Ic with IcBP occurs within the cell soon after their synthesis. The kinetics of labeling show non-coordinate processing consistent with the idea that the concentration of glycoprotein Ic limits complex formation and the subsequent processing of glycoprotein IcBP.     

Purification and characterization of a prokaryotic glucoprotein from the cell envelope of Halobacterium salinarium.

The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.     

Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding

We recently reported statistical analysis of structural data on glycosidic linkages. Here we extend this analysis to the glycan-protein linkage, and the peptide primary, secondary, and tertiary structures around N-glycosylation sites. We surveyed 506 glycoproteins in the Protein Data Bank crystallographic database, giving 2592 glycosylation sequons (1683 occupied) and generated a database of 626 nonredundant sequons with 386 occupied. Deviations in the expected amino acid composition were seen around occupied asparagines, particularly an increased occurrence of aromatic residues before the asparagine and threonine at position +2. Glycosylation alters the asparagine side chain torsion angle distribution and reduces its flexibility. There is an elevated probability of finding glycosylation sites in which secondary structure changes. An 11-class taxonomy was developed to describe protein surface geometry around glycosylation sites. Thirty-three percent of the occupied sites are on exposed convex surfaces, 10% in deep recesses and 20% on the edge of grooves with the glycan filling the cleft. A surprisingly large number of glycosylated asparagine residues have a low accessibility. The incidence of aromatic amino acids brought into close contact with the glycan by the folding process is higher than their normal levels on the surface or in the protein core. These data have significant implications for control of sequon occupancy and evolutionary selection of glycosylation sites and are discussed in relation to mechanisms of protein fold stabilization and regional quality control of protein folding. Hydrophobic protein-glycan interactions and the low accessibility of glycosylation sites in folded proteins are common features and may be critical in mediating these functions.     

Production and distribution of entactin and GP-2 in M1536-B3 cells

The endodermally differentiated mouse embryonal carcinoma cell line M1536-B3 produces the basement membrane-associated glycoproteins entactin and GP-2 in cell culture. Immunological techniques coupled with light and electron microscopy were used to study the intracellular distribution and fate of these molecules. The two proteins were localized in cisternae of rough endoplasmic reticulum and in discrete membrane-bound vesicles. There was no evidence for their presence in the Golgi apparatus. The membrane-bound vesicles appeared to fuse with the plasma membrane and thereby transfer their contents to discrete foci on the cell surface. These foci became apparent as cell-to-cell contact was made and were prominent at cell-cell contact sites. They eventually coalesced into continuous densely stained extracellular bands of amorphous material. These bands formed a honeycomb of spaces lined by cells. It appeared that production of the extracellular matrix was probably stimulated by cell-cell contact, and that cell density rather than age of the cell cultures dictated the pattern of extracellular distribution. These results support the hypothesis that these molecules are involved in cell adhesion and multicellular organization.     

Glycoproteins of trypanosomes: their biosynthesis and biological significance

1. Trypanosomes are unicellular parasites that cause human sleeping sickness in Africa and Chagas' disease in South America. Glycoproteins are important components of their plasma membrane. 2. The bloodstream form of the extracellular salivarian African trypanosome (e.g. Trypanosoma brucei) has the ability to express on its cell surface a repertoire of variant surface glycoproteins (VSGs) and in so doing, evades the immune response of the host (antigenic variation). 3. The VSG is probably synthesized initially in a manner like that of the membrane-bound glycoproteins of mammalian systems, but it also undergoes some novel post-translational modifications. 4. The stercorarian South American trypanosome (Trypanosoma cruzi) is an intracellular parasite which expresses different glycoproteins on its plasma membrane at various stages of its life-cycle, but does not exhibit antigenic variation. 5. The biosynthesis and functions of trypanosomal glycoproteins are compared with those of mammalian glycoproteins, and are discussed with particular reference to potential targets for chemotherapy and immunotherapy of trypanosomiasis.     

GALECTIN-3 expression in differentiating human myeloid cells

Galectin-3 is an endogenous mammalian carbohydrate-binding protein with affinity for ABH group carbohydrate epitopes and polylactosamine glycans present on cell surface and extracellular matrix glycoproteins. It has been shown to play a role in cell differentiation, morphogenesis, adhesion and cell proliferation regulation. Progenitor cell proliferation in bone marrow depends on stem cell factors including those modulating their adhesion to the bone marrow stroma. The present study shows that the 32 kD galectin-3 is developmentally expressed in human myeloid cells and is strongly upregulated on the cell surface of late mature myeloid cells. Despite the fact that the expression of the galectin-3 is very low in CD34+ early myeloid cell, a 70 kD protein is found by Western blotting using antibodies specific to galectin-3, exclusively in those cells. Finally, exogenous human recombinant galectin-3 binds strongly to CD34+ early myeloid cells and emphasizes granulocyte-colony stimulating factor (G-CSF) driven proliferation in vitro.     

O-mannosyl glycans in mammals

Most proteins within living organisms contain glycans. Glycan structures can modulate the biological properties and functions of glycoproteins. The major glycans of glycoproteins can be classified into two groups, N-glycans and O-glycans, according to their glycan-peptide linkage regions. Developments in glycobiology have revealed a new type of glycosidic linkage to the peptide portion, the O-mannosyl linkage, in mammals, while so far it had been thought to be specific to yeast. This review will give an outline of the O-mannosyl glycans of mammalian glycoproteins. Since one of the most well known O-mannosyl-modified mammalian glycoproteins is dystroglycan, the functional aspects of the O-mannosyl glycan of dystroglycan will be described to help understand this new glycobiological field.