The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche

The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFβ family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvβ3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFβ signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation.     

Brain-associated glycoproteins expressed in bovine heart Purkinje fibres.

The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed. Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.     

The presence of glycoproteins in the cell membrane complex of a variety of keratin fibres.

Cell membrane complex preparations have been extracted using formic acid from human hair and nail, and from the hair of sheep, alpaca, rabbit, rat, cat, and dog. On analysis they were found to have similar amino acid compositions and they all contained carbohydrate. The sugars were typical of those found in membrane glycoproteins and all preparations reacted with peroxidase-conjugated lectins.     

A histoenzymatic study of rat intrafusal muscle fibres.

The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (alpha-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K2-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkali-preincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K2-EDTA preincubation. Following either acetic acid solution or cold and room temperature K2-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K2-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing alpha-GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

Molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with rat brain synaptic functions.

The composition and biosynthesis of fucosyl glycoproteins present in rat brain synaptic membranes and synaptic junctions were investigated. Reaction with 125I-labelled fucose-binding protein (Lotus tetragonolobus) following sodium dodecyl sulphate gel electrophoresis identified 6--8 fucosyl glycoproteins in synaptic membranes but only three major high molecular classes (Mr = 180 000, 130 000 and 110 000) in synaptic junctions. Affinity chromatography on concanavalin A-Sepharose resolved each of the synaptic junctional fucosyl glycoproteins into concanavalin A-positive and negative components indicating the presence of at least six high molecular weight fucosyl glycoproteins in synaptic junctions. Following the administration of [3H]fucose synaptic membranes, synaptic junctions and post-synaptic densities incorporated isotope, the order of relative specific activities being synaptic membranes greater than synaptic junctions greater than post-synaptic densities. Fractionation of [3H]fucose-labelled synaptic junctions on concanavalin A-Sepharose revealed a time-dependent increase in the percentage of isotope associated with the concanavalin A-positive glycoproteins. The results demonstrate both molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with synaptic junctions.     

The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases.

A significant fraction of human glycosyl-phosphatidylinositol-anchored Ag CD59, CD55, CD48, and CDw52 is present in several cell lines tested (HPB-ALL, Jurkat, HL-60, Raji) in very large noncovalent complexes relatively resistant to dissociation by detergents. These complexes also contain some (glyco)lipids, such as these bearing the CD15, CDw17, and CDw65 determinants, and several intracellular components including protein tyrosine kinases and probably several of their potential substrates. Preclearing of the detergent lysates with different antibodies indicated that all these components are present jointly in a common single type of complexes the size of which is around 100 nm (molecular mass in the range of at least tens of thousands kilodaltons) as determined by ultrafiltration and gel chromatography. These results indicate the existence of cell-surface domains, specifically enriched in the above listed components, that may play a critical role in the so far poorly understood phenomenon of cell activation mediated through many different glycosyl-phosphatidylinositol-anchored (glyco)proteins and glycolipids.     

Occurrence of "elastic" fibres in the invertebrates

The connective tissues of a wide range of invertebrates have been investigated using a potassium permanganate/spirit blue staining technique. The method demonstrates fibres which are distinct from collagen, vertebrate elastic fibres, and reticular fibres. The fibres are variously associated with subepidermal collagen, muscles, basement membrances, the walls of blood vessels and the sheaths around nerve cords; the method also gives a positive reaction with the arborescent mesogloeal fibres of medusoid coelenterates. Their electron microscope appearance is distinctive and they exhibit a physical elasticity. Chemical tests indicate that following pretreatment with acidified potassium permanganate they show many, although not all, of the properties of vertebrate "elastin". It is concluded that they should be regarded as a special category of "elastic" fibre.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

Peptides of human bronchial mucus glycoproteins. Size determination by electron microscopy and by biosynthetic experiments.

Secreted human bronchial mucins, directly collected from macroscopically healthy bronchial mucosa, were prepared in the presence of six proteinase inhibitors, and analysed by electron microscopy. These mucins were similar in length distribution to molecules prepared from sputum [Slayter, Lamblin, Le Treut, Galabert, Houdret, Degand & Roussel (1984) Eur. J. Biochem. 142, 209-218], although they were a little longer, their lengths ranging up to about 1,650 nm. This length corresponds to an extended mucin peptide of about 450 kDa. In order to compare these peptide lengths with the molecular size of biosynthetic precursors, an antiserum raised against trifluoromethanesulphonic acid-treated highly glycosylated regions of human bronchial mucins was used to isolate mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling with [3H]threonine or [3H]glucosamine. A main precursor labelled with [3H]threonine and with an apparent molecular mass of about 400 kDa was detected by fluorography following SDS/polyacrylamide-gel electrophoresis. This band was observed as early as 20 min; it was more intense after a 40 min chase and had disappeared after a chase period of 280 min in unlabelled medium, presumably owing to glycosylation. Much fainter bands at about 200 kDa and between 200 and 400 kDa, also labelled with [3H]threonine, were observed mainly after a 40 min chase and had disappeared after a 280 min chase. None of these bands was labelled with [3H]glucosamine, nor did they disappear after multiple treatments with immobilized lectins. After a 280 min chase, [3H]threonine-labelled material appeared in the stacking gel, which also contained [3H]glucosamine label. The results indicate that the 200-400 kDa species are mucin precursors, whose size is comparable with that obtained by electron microscopy for respiratory mucins collected directly from the macroscopically healthy bronchial mucosa.     

Current concepts of the structure and nature of mammalian salivary mucous glycoproteins

Summary The visco-elastic properties of salivary secretions are due to high molecular-weight glyco-proteins, known as mucins. Mucins are composed of numerous oligosaccharide side-chains O-glycosidically linked through 2-acetamido-2deoxy--d-galactose to the hydroxyl groups of seryl and threonyl residues of the protein core; on the average, every fourth amino acid residue is involved in such a bond. This work conveys their isolation and purification, compiles the compositional analysis of several mammalian submaxillary and sublingual mucins; defines the conditions of the alkaline -elimination reaction, its mechanism and importance in structural studies of glycoproteins, and briefly discusses the influence of stimuli on mucous secretions, as well as biosynthesis, structural diversity, and physiological role of salivary mucous glycoproteins.     

The effects of preliminary proteolysis on the immunohistochemical and dye staining properties of elastic fibres

Summary Antibodies to -elastin peptides, amyloid P component, lysozyme and plasma protease, inhibitors have been used in an immunoperoxidase method to stain elastic fibres in frozen sections of human breast tissues. A loss of immunoreactivity seen in formalin-fixed, paraffin-embedded sections was reversed by a preliminary proteolysis. Differences in the tinctorial dye and immunohistochemical staining patterns following proteolysis by a variety of enzymes suggests a selective unmasking or removal of elastic fibre components and thus the presence of separate binding sites for individual antibodies and tinctorial dyes. Antibody blocking experiments and double immunoenzymatic labelling support the existence of several different epitopes within elastic fibres.     

Structure and expression of fibrillin-2, a novel microfibrillar component preferentially located in elastic matrices

During the previous cloning of the fibrillin gene (FBN1), we isolated a partial cDNA coding for a fibrillin-like peptide and mapped the corresponding gene (FBN2) to human chromosome 5. (Lee, B., M. Godfrey, E. Vitale, H. Hori, M. G. Mattei, M. Sarfarazi, P. Tsipouras, F. Ramirez, and D. W. Hollister. 1991. Nature [Lond.]. 352:330-334). The study left, however, unresolved whether or not the FBN2 gene product is an extracellular component structurally related to fibrillin. Work presented in this report clarifies this important point. Determination of the entire primary structure of the FBN2 gene product demonstrated that this polypeptide is highly homologous to fibrillin. Immunoelectron microscopy localized both fibrillin proteins to elastin-associated extracellular microfibrils. Finally, immunohistochemistry revealed that the fibrillins co-distribute in elastic and non-elastic connective tissues of the developing embryo, with preferential accumulation of the FBN2 gene product in elastic fiber-rich matrices. These results support the original hypothesis that the fibrillins may have distinct but related functions in the formation and maintenance of extracellular microfibrils. Accordingly, we propose to classify the FBN1 and FBN2 gene products as a new family of extracellular proteins and to name its members fibrillin-1 and fibrillin-2, respectively.