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Mucin-type glycoproteins — new perspectives on their structure and synthesis
TIBS - February 1978
38
mucin) which can be further degraded into sub-units by either disulphide bond breaking reagents or proteases. In gastric mucin, which has been thoroughly studied by Allen and co-workers [5,6], four such subunits (mol.wt. 500000) are aggregated by disulphide bonds to form the parent mucin (mol.wt. 2 x 106, Fig. 1). The disulphide bonds are located in a nonglycosylated, or naked, portion of the peptide which is protease sensitive. In tracheal mucin the native component contains a variable number of sub-units and there is good evidence that the aggregated form is dependent upon disulphide bond formation between sub-units and a crosslinking peptide [7,8]. Only the aggregated, native mucin can form gels. Mucin-mucin interactions within the gel must be highly specific to impart visco-elastic properties [9].
tion between enzymes and substrates. The glycosyltransferases are membranebound, located mainly in membranes of the smooth endoplasmic reticulum and Golgi. The maturing glycoprotein is formed within the intra-cisternal spaces of these membranes through which it moves until packaged in secretory granules [1 I]. Structural heterogeneity could arise if a particular protein-linked oligosaccharide, or nucleotide sugar-substrate failed to make contact with the appropriate transferase enzyme. Such an 'incomplete' oligosaccharide could not be further glycosylated because of the substrate specificity of the next transferase enzyme in the sequence. Of critical importance, therefore, to glycoprotein synthesis is the organisation of these transferase enzymes within membranes as this will be one of the major factors determining the integration oftransferase activities and hence the degree of completeness of the formed oligosaccharides. One must then ask the following question: "are the products of glycosyltransferase activity heterogeneous due entirely to this apparent "hit or miss" mechanism of synthesis or is there some co-ordinated scheme for the "deliberate" as well as "random" production of structural variability?" Based mainly on evidence from tracheo-bronchial mucins we will argue in favour of a 'deliberate' heterogeneity manifest both through specialisation of mucous production at the cellular level and to the presence of distinct functional and physical characteristics of the extracellular mucins.
Polydispersity and heterogeneity
lntracellular mueins
It is commonly observed that mucins have a broad molecular weight distribution indicative of considerable moleculeto-molecule variability or polydispersity [2,10], The reasons for this variability are generally considered to be the result of the biosynthetic mechanism. The protein part ofmucins is made by the messenger RNA (mRNA)-ribosome mechanism common toall proteins and is therefore under direct genetic control. The protein 'core' is the invariant part of the molecule. For the assembly of oligosaccharides to the protein there is no equivalent template to mRNA. Oligosaccharides are formed by a series of highly specific glycosyltransferases responsible for the sequential addition of monosaccharides from their nucleotide sugar precursors. These enzymes are themselves proteins and therefore of invariant structure. The frequency of glycosylation and the sequence and length of the oligosaccharide chains formed by these enzymes depends not only on their specificity but also the appropriate interac-
In mammals mucins of the tracheobronchial tract are produced by goblet cells of the surface epithelium and by submucosal glands, in which two cell types, mucous and serous, can be identified histologically. The histochemical and autoradiographic properties of mucus in these cells show that there is no randomness in the formation of mucin at the intracellular level [12,13,14]. In man and cat, gland cells produce a more sulphated mucin than goblet cells whereas the reverse is true in dogs. In man, Lamb and Reid [13] have identified on the basis of periodate-Schiff (PAS) and Alcian blue (AB) staining, four types of acidic mucin in mucous gland cells but they only appear in certain combinations in any one cell. This segregation and selectivity in mucin synthesis presumably reflects differences in the organisation of transferase enzymes and/or, specific enzyme activation/repression phenomena. Additional evidence of a chemical distinction between submucosal gland and goblet cell mucins has been obtained by
Mucin-type glycoproteinsnew perspectives on their structure and synthesis J.T. Gallagher and A.P. Corfield The visco-elastic properties of mucous secretions are due to mucin-tjTe glycoproteins whose structural variability may indicate the presence of distinct glycoprotein populations rather than a continuous series of related structures.
Glycoproteins are complex macromolecules whose structural and functional roles reside mainly in the extracellular phase or as components of cell membranes. We are concerned here with glycoproteins found in mucous secretions, particularly mucus produced by the tracheo-bronchial tract. Mucous glycoproteins are called mucins and are responsible for the viscous and gelforming properties of mucus [1,2]. In contrast to other glycoproteins, mucins are rich in carbohydrate (70-85'!, of dry weight) and the protein core region has a characteristic amino acid composition in which serine, threonine, proline and glycine predominate [2,3,4]. The linkage between protein and carbohydrate is an Oglycosidic one between serine or threonine and N-acetylgalactosamine (Fig. I ). In tracheo-bronchial mucins there is, on average, one oligosaccharide chain for every 4-6 amino acids. Mucins are negatively charged due to the presence of sialic acid at the terminal, non-reducing end of some of the oligosaccharide chains and to the frequent, but not universal presence of ester sulphate residues also located on the carbohydrate portion of the molecule. Fucose is the other most common t~rminal sugar but in some mucins, which possess ABO blood group activity, x-linked galactose or N-acetylgalactosamine are at chain termini. Most mammalian mucins do not contain mannose or uronic acid.
Sub-unit structure and gel formation Mucous gels can be dissolved by strong denaturing agents, such as 6 M urea or 4.0 M guanidine hydrochloride, which do not break covalent bonds. The main product is a solution of high molecular weight glycoprotein (native or parent J. T.G. is at the Cancer Research Campaign Department of Medical Oacology, Christie Hospital and Holt Radium Institute. Manchester M20 9BX, U.K. A.P.C. is at the Biochenlische hlstitut im Fachbereich Medizin, Christian-Albrecht-Universitiit, 23, Kiel, G.F.R,
39
T I B S - February 1978
a technique for selectively stimulating secretions from these two cell types [15]. Secreted gland cell mucin was enriched in sulphate whereas goblet cell mucin showed reduced sulphation but sialic acid levels were increased at the expense of fucose. Since most of the biochemical studies on tracheo-bronchial and other mucins are carried out on either secreted mucins or whole gland extracts, the problem arises of detecting specific forms of macromolecular organisation or differentiated functions.
Specific sub-unit aggregation It has recently been shown that when mucins are collected from the perfused cat trachea in situ or from rabbit trachea in organ culture, they can be readily dissolved by the addition of 6 M urea to yield the native mucin [15,16]. Fractionation by DEAE-ion exchange chromatography and examination ofeluted material by electrophorcsis has shown that mucins can be separated into a range of molecular types from PAS-positive, AB-negative 'neutral'
mucins of low mobility to more mobile components stainable only with AB. Between these two extremes, the mucins stain with both PAS and AB. In cat mucins we also have evidence from [35S]sulphate and pH]glucosc labelling patterns that the more acidic the mucin (AB-positive only) the more rapid the synthesis and the less the heterogeneity [I 6]. From these results, it appears that there is no random mixing of mucins of differing acidities and metabolic activities. The form of sub-unit aggregation in the native mucin in the
O•o ~~.,.R SUGAR ESIDUES...j I ~1~ SH
J-r--
GLYCOSYLATED PORTION
'1".... l
OXIDATION
SH REDUCTION NAKED PEPTIDE
MUCIN SUBUNIT(n)
rx r 4 t
©
=
-0
-?
MUC]N
HIGH CONCENTRATIONS
~t TRACHEAL ~OENATURATION "."~~ 5i" MUCIN ,:
NATIVE MUCIN
•
d
4'
GASTRIC
GEL
..
~'~/t ((
Fig. 1. Levels of organisation in mucins - bigger and 'stickier '. Sub-units are aggregated either by direct disulphide bond formation with other sub-units (gastric mucin) or through cross-linking peptides (tracheal mucin) to give the native mucin which can form gels at high concentration.
40 extracellular phase may therefore maintain the segregation of mucins observed at the intracellular level. Such a mechanism would favour the clustering of sub-units of similar functional properties. This concept prompts the question of at what point in the temporal sequence of synthesis, storage and secretion does aggregation of sub-units, which are presumed to be the primary biosynthetic products, occur. It is attractive to speculate that mucins are stored in cells in their reduced, disaggregated form which would preclude a gel structure. Disulphide bond formation at the time of secretion would yield the native, gel-forming mucin (Fig. 1).
Mucin sub-units in sputum Sputum is a pathological secretion of the mammalian tracheo-bronchial tract and is rich in mucin. Havez and co-workers [17,18] have shown that mucin sub-units from human sputum can be separated into three classes, the neutral 'fucomucins' and the acidic 'sulpho-' and 'sialomucins'. They found important differences in the biological and physical properties of these mucins; for example, only the neutral fraction possessed blood group activity whereas the sialomucins were most important in gel formation [19]. Sialomucins were also the only class of mucin able to inhibit bradykinin-induced bronchospasm in rabbits. These results strongly support the concept of the synthesis of specific types of mucin rather than a continuously variable population. Some of the findings of the Havez group have been challenged by Boat and coworkers [20] and Roberts [21] who both found blood group activity in strong and weak acidic mucins. However, an interesting finding from the Boat group was that only the more acidic mucins could inhibit influenza virus hemagglutination [20]. Activity was dependent upon neuraminidasesensitive sialic acid residues and this result underlines an important biological distinction between acidic and neutral mucins.
Tight regulation throughout mucin biosynthesis A potential source of heterogeneity in mucins would be an erratic, poorly controlled supply of 'activated' nucleotide sugars required for glycosylation. The available evidence suggests, however, that levels of sugar precursors are tightly regulated; the metabolic pathways leading to the nucleotide sugars are controlled at the substrate level by feedback mechanisms where the finalproduct inhibits the first enzyme in the pathway. The priming of amino-sugars and sialic acids serves as an example where the effector moderated
T I B S - F e b r u a r y 1978
feedback mechanisms involving glucosamine-6-phosphate synthetase (aminosugars and sialic acids) and UDP-Nacetylglucosamine 2' epimerase (sialic acids), ubiquitous in mammalian glycoprotein syrithesising tissues, have been adapted to suit the tracheal system [22,23]. In addition, there are several other important controls, notably the equilibrium of the phosphorylase reactions leading directly to the nucleotide sugars, the availability of substrates and the influence of effectors and hormones on individual enzymes. This organised regulation is clearly designed to control the rate of formation, identity and energy utilisation during mucin biosynthesis. The problem of how the nucleotide sugars interact with membrane-bound glycosyltransferase is unclear and in contrast to the biosynthesis of N-glycosyllinked glycoproteins, no lipid intermedia: tes have been implicated in the O-glycosyllinked mucins. The specific addition of monosaccharides and sulphate by transferase complexes in membranes is a second site of regulation of synthesis. The susceptibility of transferase enzymes to control by substrate levels or by hormones and effectors [24] will determine the nature of the completed mucin. A further feature of glycoprotein biosynthetic regulation is the physical separation of individua! reactions within the cell. The activation of sugars is cytoplasmic with the important exception of specific steps in the activation of the sialic acids [25]. All nucleotide sugars are subsequently utilised in membrane systems, calling for integration of cytosol and membrane pools. The flexibility of the overall regulation is emphasised by the maintenance of adenylate charge potential and of cytosine, guanine and uridine triphosphate pools, required for nucleotide sugar synthesis, even after prolonged maximal secretion rates [26].
Other aspects of mucin specificity Purified, native, gastric mucin will bind to monolayers ofcultured cells (e.g., BHK, HeLa); mucin binding inhibits the adhesion to the monolayer of cell suspensions of a similar or different cell type [27]. However, despite the purity of the mucin preparation [28] only about 6"0 of the mucin possessed the requisite structure to combine with cell surface binding sites. Thus, a small proportion of the mucin molecules differ from the bulk material and their cell adhesion properties could be of considerable physiological importance in view of the 'barrier' function of gastric mucus. Examples of specificity of synthesis have come from studies on bronchogenic cysts.
These are developmental abnormalities of the tracheo-bronchial epithelium in which a small number of cells bud off from the main airway lining and eventually form cysts. Mucus produced by these cysts is not usually representative of mucus formed in the whole respiratory tract and often only one type ofmucin (neutral, sialo or sulphomucin) is produced [29]. Since the histochemical evidence discussed previously shows a non-random distribution of intracellular mucins in the trachea, it is quite predictable that the selected cell population in bronchogenic cysts would be restricted in the range of mucin types that they can produce. These findings confirm that there is differentiation at the cellular level in terms of biosynthetic capacity and that variability of extracellular mucin is not entirely the result of randomness in biosynthesis. To conclude, we feel that the foregoing evidence suggests that the well-documented variation in mucin-type glycoproteins may be of a more complex and subtle form than is generally appreciated, particularly in the tracheo-bronchial mucins. These mucins consist of distinct populations of molecules and although within any particular population, considerable heterogeneity can occur [30], there are sufficient distinguishing characteristics to envisage respiratory mucins as representing an example of discontinuous rather than continuous polydispersity [10].
Acknowledgement J.T.G. thanks The Cystic Fibrosis Research Foundation Trust for financial support.
References 1 Allen,A., Pain, R. H. and Robinson,T. R. (1976) Nature (London) 264, 88-89 2 Gibbons, R.A. and Sellwood, R. (1973) in The Biology of the Cervix (Blandau, R.J. and Moghissi, K., eds)pp. 251-265, Universityof Chicago Press, Chicago 3 Gottscbalk, A. and Bhargava. A.S. (1972) in Glrcoprotehls (Gottschalk, A., ed.) pp. 810-829, Elsevier, Amsterdam 4 Meyer, F.A. (1976)Biorheology 13, 49-58 5 Allen,A., Pain, R. H. and Snary, D. (1974) Faraday Discussions 57, 210-220 6 Scawen, M. and Allen,A. (1977)Biochem. J. 163, 363-368 7 Creeth,J. M., Bhaskar,K. R., Horton,J. R., Das, I., Lopez-Vidriero,M.T. and Reid, L. (1977)Biochem. J. in press 8 Roberts, G.P. (1976) Archs. Biochem. Biophvs. 173, 528 537 9 Allen, A. (1977) in Mucus ha Health and Disease (Parke, D.V. and Elstein,M., eds) Plenum Press, N.Y. in press 10 Gibbons, R.A. (1972) in Glycoprotehas (Gottschalk, A., ed.) pp. 31-140, Elsevier,Amsterdam I I Meyrick,B. and Reid, L. (1975)J. Cell. BioL 67, 320-344 12 Gallagher, J.T., Kent, P.W., Passatore, M., Phipps, R.J., Richardson, P.S. and Lamb, D. (1975) Proc. R. Soc. Lond. B. 192,49-76
41
T I B S - February 1978 13 Lamb, D. and Reid, L. (1969) J. Path. 98, 213-229 14 Spicer, S.S,, Chakrin, L.W., Wardell, J.R. and Kendrick, W. (1971) Lab. hlvest. 25, 483490 15 Gallagher, J.T., Kent, P.W., Phipps. R.J. and Richardson, P.S. (1977) in Mucus hi Health and Disease (Parke, D.V. and Elstein. M.. eds) Plenum Press. N.Y. in press 16 Gallagher, J.T. (Unpublished observations) 17 Havez, R., Roussel, P., Regand, P., Randoux. A. and Biserte, G. (1969) in Protides of the Biological Fhdds (Peeters, H.. ed.) pp. 343-360, Pergamon Press, London 18 Havez, R., Roussel, P., Degand. P. and Biserte, G. (1967) Clin. Chhn. Acta 17, 463-477 19 Puchelle. E.. Zahm, J.M. and Havez, R. (1973) Ball Physio-path. Resp. 9, 237-256 20 Boat. T.F., Chang, P.W., lyer, R.N., Carlson, D. M. and Polony, I. (1976) Archs. Biochem. Bigphys. 177.95-104 21 Roberts, G.P. (1974) Eur. J. Biochem. 50, 265 280 22 Ellis, D.B.. Munro, J. R. and Stahl, G. H. (1972) Biochhn. Biophys. Acta 289, 108-116
23 Ellis, D.B. and Sommar, K.M. (1972) Biochim. Biophys. Acta 276, 105-112 24 Baker, A. P.. Hillegass, C. M., Holden, D.A. and Smith, W.J. (1977) Am. Rev. Resp. Dis. I 15, 811817 25 Corfield, A. P., Ferreira do Amaral, C., Wember, M. and Schauer, R. (1976) Ear. J. Biochem. 68. 597-610 26 Phelps, C. F. and Young, A. M. (1977) in Mncus ht Health mid Disease (Parke. D.V. and Elstein, IVI.,eds) Plenum Press. N.Y. in press 27 Allen, A. and Megan Minnikin, S. (1975) J. Cell Sci. 17, 617-631 28 Starkey, B.J., Snary, D. and Allen. A. (1974) Bigchem. J. 141,633-639 29 Degand, P.. Roussel, P.. Lamblin, G. and Havez, R. (1973) Biochim. Biophys. Acta 320, 318-330 30 Roussel, P., Lamblin, G., Degand, P., WalkerNasir, E. and Jeanloz, R. W. (1975) J. Biol. Chem. 250, 2114-2122
Ribosome structure studies by low angle neutron scattering R. Parfait, M.H.J. Koch, H.B. Stuhrmann and R.R. Crichton Low angle neutron scattering shows that 50 S and 70 S ribosomal particles have their RNA located mostly towards the insid& The 30 S is more homogeneous. A low resolution shape is proposed for the 50 S subunit. Structure determination for large macromolecular complexes such as ribosomes, fatty acid synthetase or RNA polymerase poses a number of special problems. The application of classical methods requires suitable crystals, the unit cell dimensions are large and the degree of structural order may not be sufficient to permit structure determination at high resolution. Thus a number of other approaches have been adopted to analyse the structure of such complexes. As we shall see these approaches have their advantages as well as their disadvantages. In studies on ribosomes the greatest progress to date has been made using electron microscopy of negatively stained ribosomal preparations. Not only have Georg StSffler and his colleagues in Berlin [1] and Jim Lake in Los Angeles I-2] presented R.P. and R.R.C. are at the Unitb de Biochimie, Universitb Catholique de Louvain, Place Louis Pasteur l, B-1348 Louvain-la-Neuve, Belgium. M.H.J.K. and H.B.S. are at EMBL c/o DES}', 2000 Hamburg 52, Notkesteig I, G.F.R.
three-dimensional models of the 30 S and 50 S subunits of Escherichia coli ribosomes, they are also well advanced in the localisation of most of the 21 ribosomal proteins of the small subunit and many of the 34 proteins of the large subunit by electron microscopy of ribosomal subunits cross-linked by antibodies directed against each of the individual ribosomal proteins. We will return to a discussion of these models later: for the moment it should be pointed out that these models represent three-dimensional reconstructions of twodimensional transmission images obtained with vacuum dried ribosomal particles which have been embedded on carboncoated grids and visualised by negative shadowing with heavy metal salts. A second group of techniques which promises to give useful data concerning ribosomal structure are low angle scattering methods using X-rays or neutrons, and it is with the latter that we will be concerned here. These techniques have the great advantage of being non-destructive and applicable to aqueous solutions of the
biological macromolecules. Thus, for example, the ability of ribosomal particles to synthesise protein can be tested both before and after the scattering measurements. The major disadvantage of low angle scattering techniques is that structure analysis cannot be carried to high resolution. However, as we will show, the use of neutrons allows us to obtain information about the distribution of protein and RNA in the ribosome, which cannot be obtained by X-ray scattering or by electron microscopy. Low angle neutron scattering Neutrons, like electrons, have wave properties. Indeed, thermal neutrons have wavelengths of the order of I A (10 -a cm) and so are suitable for studies at atomic resolution. The design of the neutron low angle scattering device used for biological samples at the lnstitut Max von Laue-Paul Langevin, Grenoble is illustrated in Fig. 1. The neutrons are derived from the fission of uranium in the core of the nuclear reactor; their kinetic energy is much too great for biological studies, and so they are slowed down by a 'cold source' consisting at Grenoble of 25 1 of liquid deuterium cooled to around 25°K. This shifts the wavelength of the neutron beam to around 6 A.. The flux of cold neutrons is then guided to a mechanical velocity selector which enables a homogeneous beam of neutrons of a defined wavelength (usually between 5-8 A) to be obtained. The neutron beam is collimated and impinges on the sample: neutrons that are
E]
reactor
core
cold source (D2] neutron guide velocity selector
neutron guides,removable [collimation)
rlsample
[~j
detector,variable positon computer ( controbstorage)
Fig. I. Experimental set-up for a neutron low angle scattering experiment.
38
mucin) which can be further degraded into sub-units by either disulphide bond breaking reagents or proteases. In gastric mucin, which has been thoroughly studied by Allen and co-workers [5,6], four such subunits (mol.wt. 500000) are aggregated by disulphide bonds to form the parent mucin (mol.wt. 2 x 106, Fig. 1). The disulphide bonds are located in a nonglycosylated, or naked, portion of the peptide which is protease sensitive. In tracheal mucin the native component contains a variable number of sub-units and there is good evidence that the aggregated form is dependent upon disulphide bond formation between sub-units and a crosslinking peptide [7,8]. Only the aggregated, native mucin can form gels. Mucin-mucin interactions within the gel must be highly specific to impart visco-elastic properties [9].
tion between enzymes and substrates. The glycosyltransferases are membranebound, located mainly in membranes of the smooth endoplasmic reticulum and Golgi. The maturing glycoprotein is formed within the intra-cisternal spaces of these membranes through which it moves until packaged in secretory granules [1 I]. Structural heterogeneity could arise if a particular protein-linked oligosaccharide, or nucleotide sugar-substrate failed to make contact with the appropriate transferase enzyme. Such an 'incomplete' oligosaccharide could not be further glycosylated because of the substrate specificity of the next transferase enzyme in the sequence. Of critical importance, therefore, to glycoprotein synthesis is the organisation of these transferase enzymes within membranes as this will be one of the major factors determining the integration oftransferase activities and hence the degree of completeness of the formed oligosaccharides. One must then ask the following question: "are the products of glycosyltransferase activity heterogeneous due entirely to this apparent "hit or miss" mechanism of synthesis or is there some co-ordinated scheme for the "deliberate" as well as "random" production of structural variability?" Based mainly on evidence from tracheo-bronchial mucins we will argue in favour of a 'deliberate' heterogeneity manifest both through specialisation of mucous production at the cellular level and to the presence of distinct functional and physical characteristics of the extracellular mucins.
Polydispersity and heterogeneity
lntracellular mueins
It is commonly observed that mucins have a broad molecular weight distribution indicative of considerable moleculeto-molecule variability or polydispersity [2,10], The reasons for this variability are generally considered to be the result of the biosynthetic mechanism. The protein part ofmucins is made by the messenger RNA (mRNA)-ribosome mechanism common toall proteins and is therefore under direct genetic control. The protein 'core' is the invariant part of the molecule. For the assembly of oligosaccharides to the protein there is no equivalent template to mRNA. Oligosaccharides are formed by a series of highly specific glycosyltransferases responsible for the sequential addition of monosaccharides from their nucleotide sugar precursors. These enzymes are themselves proteins and therefore of invariant structure. The frequency of glycosylation and the sequence and length of the oligosaccharide chains formed by these enzymes depends not only on their specificity but also the appropriate interac-
In mammals mucins of the tracheobronchial tract are produced by goblet cells of the surface epithelium and by submucosal glands, in which two cell types, mucous and serous, can be identified histologically. The histochemical and autoradiographic properties of mucus in these cells show that there is no randomness in the formation of mucin at the intracellular level [12,13,14]. In man and cat, gland cells produce a more sulphated mucin than goblet cells whereas the reverse is true in dogs. In man, Lamb and Reid [13] have identified on the basis of periodate-Schiff (PAS) and Alcian blue (AB) staining, four types of acidic mucin in mucous gland cells but they only appear in certain combinations in any one cell. This segregation and selectivity in mucin synthesis presumably reflects differences in the organisation of transferase enzymes and/or, specific enzyme activation/repression phenomena. Additional evidence of a chemical distinction between submucosal gland and goblet cell mucins has been obtained by
Mucin-type glycoproteinsnew perspectives on their structure and synthesis J.T. Gallagher and A.P. Corfield The visco-elastic properties of mucous secretions are due to mucin-tjTe glycoproteins whose structural variability may indicate the presence of distinct glycoprotein populations rather than a continuous series of related structures.
Glycoproteins are complex macromolecules whose structural and functional roles reside mainly in the extracellular phase or as components of cell membranes. We are concerned here with glycoproteins found in mucous secretions, particularly mucus produced by the tracheo-bronchial tract. Mucous glycoproteins are called mucins and are responsible for the viscous and gelforming properties of mucus [1,2]. In contrast to other glycoproteins, mucins are rich in carbohydrate (70-85'!, of dry weight) and the protein core region has a characteristic amino acid composition in which serine, threonine, proline and glycine predominate [2,3,4]. The linkage between protein and carbohydrate is an Oglycosidic one between serine or threonine and N-acetylgalactosamine (Fig. I ). In tracheo-bronchial mucins there is, on average, one oligosaccharide chain for every 4-6 amino acids. Mucins are negatively charged due to the presence of sialic acid at the terminal, non-reducing end of some of the oligosaccharide chains and to the frequent, but not universal presence of ester sulphate residues also located on the carbohydrate portion of the molecule. Fucose is the other most common t~rminal sugar but in some mucins, which possess ABO blood group activity, x-linked galactose or N-acetylgalactosamine are at chain termini. Most mammalian mucins do not contain mannose or uronic acid.
Sub-unit structure and gel formation Mucous gels can be dissolved by strong denaturing agents, such as 6 M urea or 4.0 M guanidine hydrochloride, which do not break covalent bonds. The main product is a solution of high molecular weight glycoprotein (native or parent J. T.G. is at the Cancer Research Campaign Department of Medical Oacology, Christie Hospital and Holt Radium Institute. Manchester M20 9BX, U.K. A.P.C. is at the Biochenlische hlstitut im Fachbereich Medizin, Christian-Albrecht-Universitiit, 23, Kiel, G.F.R,
39
T I B S - February 1978
a technique for selectively stimulating secretions from these two cell types [15]. Secreted gland cell mucin was enriched in sulphate whereas goblet cell mucin showed reduced sulphation but sialic acid levels were increased at the expense of fucose. Since most of the biochemical studies on tracheo-bronchial and other mucins are carried out on either secreted mucins or whole gland extracts, the problem arises of detecting specific forms of macromolecular organisation or differentiated functions.
Specific sub-unit aggregation It has recently been shown that when mucins are collected from the perfused cat trachea in situ or from rabbit trachea in organ culture, they can be readily dissolved by the addition of 6 M urea to yield the native mucin [15,16]. Fractionation by DEAE-ion exchange chromatography and examination ofeluted material by electrophorcsis has shown that mucins can be separated into a range of molecular types from PAS-positive, AB-negative 'neutral'
mucins of low mobility to more mobile components stainable only with AB. Between these two extremes, the mucins stain with both PAS and AB. In cat mucins we also have evidence from [35S]sulphate and pH]glucosc labelling patterns that the more acidic the mucin (AB-positive only) the more rapid the synthesis and the less the heterogeneity [I 6]. From these results, it appears that there is no random mixing of mucins of differing acidities and metabolic activities. The form of sub-unit aggregation in the native mucin in the
O•o ~~.,.R SUGAR ESIDUES...j I ~1~ SH
J-r--
GLYCOSYLATED PORTION
'1".... l
OXIDATION
SH REDUCTION NAKED PEPTIDE
MUCIN SUBUNIT(n)
rx r 4 t
©
=
-0
-?
MUC]N
HIGH CONCENTRATIONS
~t TRACHEAL ~OENATURATION "."~~ 5i" MUCIN ,:
NATIVE MUCIN
•
d
4'
GASTRIC
GEL
..
~'~/t ((
Fig. 1. Levels of organisation in mucins - bigger and 'stickier '. Sub-units are aggregated either by direct disulphide bond formation with other sub-units (gastric mucin) or through cross-linking peptides (tracheal mucin) to give the native mucin which can form gels at high concentration.
40 extracellular phase may therefore maintain the segregation of mucins observed at the intracellular level. Such a mechanism would favour the clustering of sub-units of similar functional properties. This concept prompts the question of at what point in the temporal sequence of synthesis, storage and secretion does aggregation of sub-units, which are presumed to be the primary biosynthetic products, occur. It is attractive to speculate that mucins are stored in cells in their reduced, disaggregated form which would preclude a gel structure. Disulphide bond formation at the time of secretion would yield the native, gel-forming mucin (Fig. 1).
Mucin sub-units in sputum Sputum is a pathological secretion of the mammalian tracheo-bronchial tract and is rich in mucin. Havez and co-workers [17,18] have shown that mucin sub-units from human sputum can be separated into three classes, the neutral 'fucomucins' and the acidic 'sulpho-' and 'sialomucins'. They found important differences in the biological and physical properties of these mucins; for example, only the neutral fraction possessed blood group activity whereas the sialomucins were most important in gel formation [19]. Sialomucins were also the only class of mucin able to inhibit bradykinin-induced bronchospasm in rabbits. These results strongly support the concept of the synthesis of specific types of mucin rather than a continuously variable population. Some of the findings of the Havez group have been challenged by Boat and coworkers [20] and Roberts [21] who both found blood group activity in strong and weak acidic mucins. However, an interesting finding from the Boat group was that only the more acidic mucins could inhibit influenza virus hemagglutination [20]. Activity was dependent upon neuraminidasesensitive sialic acid residues and this result underlines an important biological distinction between acidic and neutral mucins.
Tight regulation throughout mucin biosynthesis A potential source of heterogeneity in mucins would be an erratic, poorly controlled supply of 'activated' nucleotide sugars required for glycosylation. The available evidence suggests, however, that levels of sugar precursors are tightly regulated; the metabolic pathways leading to the nucleotide sugars are controlled at the substrate level by feedback mechanisms where the finalproduct inhibits the first enzyme in the pathway. The priming of amino-sugars and sialic acids serves as an example where the effector moderated
T I B S - F e b r u a r y 1978
feedback mechanisms involving glucosamine-6-phosphate synthetase (aminosugars and sialic acids) and UDP-Nacetylglucosamine 2' epimerase (sialic acids), ubiquitous in mammalian glycoprotein syrithesising tissues, have been adapted to suit the tracheal system [22,23]. In addition, there are several other important controls, notably the equilibrium of the phosphorylase reactions leading directly to the nucleotide sugars, the availability of substrates and the influence of effectors and hormones on individual enzymes. This organised regulation is clearly designed to control the rate of formation, identity and energy utilisation during mucin biosynthesis. The problem of how the nucleotide sugars interact with membrane-bound glycosyltransferase is unclear and in contrast to the biosynthesis of N-glycosyllinked glycoproteins, no lipid intermedia: tes have been implicated in the O-glycosyllinked mucins. The specific addition of monosaccharides and sulphate by transferase complexes in membranes is a second site of regulation of synthesis. The susceptibility of transferase enzymes to control by substrate levels or by hormones and effectors [24] will determine the nature of the completed mucin. A further feature of glycoprotein biosynthetic regulation is the physical separation of individua! reactions within the cell. The activation of sugars is cytoplasmic with the important exception of specific steps in the activation of the sialic acids [25]. All nucleotide sugars are subsequently utilised in membrane systems, calling for integration of cytosol and membrane pools. The flexibility of the overall regulation is emphasised by the maintenance of adenylate charge potential and of cytosine, guanine and uridine triphosphate pools, required for nucleotide sugar synthesis, even after prolonged maximal secretion rates [26].
Other aspects of mucin specificity Purified, native, gastric mucin will bind to monolayers ofcultured cells (e.g., BHK, HeLa); mucin binding inhibits the adhesion to the monolayer of cell suspensions of a similar or different cell type [27]. However, despite the purity of the mucin preparation [28] only about 6"0 of the mucin possessed the requisite structure to combine with cell surface binding sites. Thus, a small proportion of the mucin molecules differ from the bulk material and their cell adhesion properties could be of considerable physiological importance in view of the 'barrier' function of gastric mucus. Examples of specificity of synthesis have come from studies on bronchogenic cysts.
These are developmental abnormalities of the tracheo-bronchial epithelium in which a small number of cells bud off from the main airway lining and eventually form cysts. Mucus produced by these cysts is not usually representative of mucus formed in the whole respiratory tract and often only one type ofmucin (neutral, sialo or sulphomucin) is produced [29]. Since the histochemical evidence discussed previously shows a non-random distribution of intracellular mucins in the trachea, it is quite predictable that the selected cell population in bronchogenic cysts would be restricted in the range of mucin types that they can produce. These findings confirm that there is differentiation at the cellular level in terms of biosynthetic capacity and that variability of extracellular mucin is not entirely the result of randomness in biosynthesis. To conclude, we feel that the foregoing evidence suggests that the well-documented variation in mucin-type glycoproteins may be of a more complex and subtle form than is generally appreciated, particularly in the tracheo-bronchial mucins. These mucins consist of distinct populations of molecules and although within any particular population, considerable heterogeneity can occur [30], there are sufficient distinguishing characteristics to envisage respiratory mucins as representing an example of discontinuous rather than continuous polydispersity [10].
Acknowledgement J.T.G. thanks The Cystic Fibrosis Research Foundation Trust for financial support.
References 1 Allen,A., Pain, R. H. and Robinson,T. R. (1976) Nature (London) 264, 88-89 2 Gibbons, R.A. and Sellwood, R. (1973) in The Biology of the Cervix (Blandau, R.J. and Moghissi, K., eds)pp. 251-265, Universityof Chicago Press, Chicago 3 Gottscbalk, A. and Bhargava. A.S. (1972) in Glrcoprotehls (Gottschalk, A., ed.) pp. 810-829, Elsevier, Amsterdam 4 Meyer, F.A. (1976)Biorheology 13, 49-58 5 Allen,A., Pain, R. H. and Snary, D. (1974) Faraday Discussions 57, 210-220 6 Scawen, M. and Allen,A. (1977)Biochem. J. 163, 363-368 7 Creeth,J. M., Bhaskar,K. R., Horton,J. R., Das, I., Lopez-Vidriero,M.T. and Reid, L. (1977)Biochem. J. in press 8 Roberts, G.P. (1976) Archs. Biochem. Biophvs. 173, 528 537 9 Allen, A. (1977) in Mucus ha Health and Disease (Parke, D.V. and Elstein,M., eds) Plenum Press, N.Y. in press 10 Gibbons, R.A. (1972) in Glycoprotehas (Gottschalk, A., ed.) pp. 31-140, Elsevier,Amsterdam I I Meyrick,B. and Reid, L. (1975)J. Cell. BioL 67, 320-344 12 Gallagher, J.T., Kent, P.W., Passatore, M., Phipps, R.J., Richardson, P.S. and Lamb, D. (1975) Proc. R. Soc. Lond. B. 192,49-76
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T I B S - February 1978 13 Lamb, D. and Reid, L. (1969) J. Path. 98, 213-229 14 Spicer, S.S,, Chakrin, L.W., Wardell, J.R. and Kendrick, W. (1971) Lab. hlvest. 25, 483490 15 Gallagher, J.T., Kent, P.W., Phipps. R.J. and Richardson, P.S. (1977) in Mucus hi Health and Disease (Parke, D.V. and Elstein. M.. eds) Plenum Press. N.Y. in press 16 Gallagher, J.T. (Unpublished observations) 17 Havez, R., Roussel, P., Regand, P., Randoux. A. and Biserte, G. (1969) in Protides of the Biological Fhdds (Peeters, H.. ed.) pp. 343-360, Pergamon Press, London 18 Havez, R., Roussel, P., Degand. P. and Biserte, G. (1967) Clin. Chhn. Acta 17, 463-477 19 Puchelle. E.. Zahm, J.M. and Havez, R. (1973) Ball Physio-path. Resp. 9, 237-256 20 Boat. T.F., Chang, P.W., lyer, R.N., Carlson, D. M. and Polony, I. (1976) Archs. Biochem. Bigphys. 177.95-104 21 Roberts, G.P. (1974) Eur. J. Biochem. 50, 265 280 22 Ellis, D.B.. Munro, J. R. and Stahl, G. H. (1972) Biochhn. Biophys. Acta 289, 108-116
23 Ellis, D.B. and Sommar, K.M. (1972) Biochim. Biophys. Acta 276, 105-112 24 Baker, A. P.. Hillegass, C. M., Holden, D.A. and Smith, W.J. (1977) Am. Rev. Resp. Dis. I 15, 811817 25 Corfield, A. P., Ferreira do Amaral, C., Wember, M. and Schauer, R. (1976) Ear. J. Biochem. 68. 597-610 26 Phelps, C. F. and Young, A. M. (1977) in Mncus ht Health mid Disease (Parke. D.V. and Elstein, IVI.,eds) Plenum Press. N.Y. in press 27 Allen, A. and Megan Minnikin, S. (1975) J. Cell Sci. 17, 617-631 28 Starkey, B.J., Snary, D. and Allen. A. (1974) Bigchem. J. 141,633-639 29 Degand, P.. Roussel, P.. Lamblin, G. and Havez, R. (1973) Biochim. Biophys. Acta 320, 318-330 30 Roussel, P., Lamblin, G., Degand, P., WalkerNasir, E. and Jeanloz, R. W. (1975) J. Biol. Chem. 250, 2114-2122
Ribosome structure studies by low angle neutron scattering R. Parfait, M.H.J. Koch, H.B. Stuhrmann and R.R. Crichton Low angle neutron scattering shows that 50 S and 70 S ribosomal particles have their RNA located mostly towards the insid& The 30 S is more homogeneous. A low resolution shape is proposed for the 50 S subunit. Structure determination for large macromolecular complexes such as ribosomes, fatty acid synthetase or RNA polymerase poses a number of special problems. The application of classical methods requires suitable crystals, the unit cell dimensions are large and the degree of structural order may not be sufficient to permit structure determination at high resolution. Thus a number of other approaches have been adopted to analyse the structure of such complexes. As we shall see these approaches have their advantages as well as their disadvantages. In studies on ribosomes the greatest progress to date has been made using electron microscopy of negatively stained ribosomal preparations. Not only have Georg StSffler and his colleagues in Berlin [1] and Jim Lake in Los Angeles I-2] presented R.P. and R.R.C. are at the Unitb de Biochimie, Universitb Catholique de Louvain, Place Louis Pasteur l, B-1348 Louvain-la-Neuve, Belgium. M.H.J.K. and H.B.S. are at EMBL c/o DES}', 2000 Hamburg 52, Notkesteig I, G.F.R.
three-dimensional models of the 30 S and 50 S subunits of Escherichia coli ribosomes, they are also well advanced in the localisation of most of the 21 ribosomal proteins of the small subunit and many of the 34 proteins of the large subunit by electron microscopy of ribosomal subunits cross-linked by antibodies directed against each of the individual ribosomal proteins. We will return to a discussion of these models later: for the moment it should be pointed out that these models represent three-dimensional reconstructions of twodimensional transmission images obtained with vacuum dried ribosomal particles which have been embedded on carboncoated grids and visualised by negative shadowing with heavy metal salts. A second group of techniques which promises to give useful data concerning ribosomal structure are low angle scattering methods using X-rays or neutrons, and it is with the latter that we will be concerned here. These techniques have the great advantage of being non-destructive and applicable to aqueous solutions of the
biological macromolecules. Thus, for example, the ability of ribosomal particles to synthesise protein can be tested both before and after the scattering measurements. The major disadvantage of low angle scattering techniques is that structure analysis cannot be carried to high resolution. However, as we will show, the use of neutrons allows us to obtain information about the distribution of protein and RNA in the ribosome, which cannot be obtained by X-ray scattering or by electron microscopy. Low angle neutron scattering Neutrons, like electrons, have wave properties. Indeed, thermal neutrons have wavelengths of the order of I A (10 -a cm) and so are suitable for studies at atomic resolution. The design of the neutron low angle scattering device used for biological samples at the lnstitut Max von Laue-Paul Langevin, Grenoble is illustrated in Fig. 1. The neutrons are derived from the fission of uranium in the core of the nuclear reactor; their kinetic energy is much too great for biological studies, and so they are slowed down by a 'cold source' consisting at Grenoble of 25 1 of liquid deuterium cooled to around 25°K. This shifts the wavelength of the neutron beam to around 6 A.. The flux of cold neutrons is then guided to a mechanical velocity selector which enables a homogeneous beam of neutrons of a defined wavelength (usually between 5-8 A) to be obtained. The neutron beam is collimated and impinges on the sample: neutrons that are
E]
reactor
core
cold source (D2] neutron guide velocity selector
neutron guides,removable [collimation)
rlsample
[~j
detector,variable positon computer ( controbstorage)
Fig. I. Experimental set-up for a neutron low angle scattering experiment.