British Medical Bulletin (1978) Vol. 34, No. 1, pp. 5-8
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp
THE BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS LYNNE REED* MD FRCP FRCPath Harvard Medical School Boston, Massachusetts, USA and The Children's Hospital Medical Center Boston, Massachusetts, USA
J R CLAMP MD PhD FRIC MRCP Clinical Research Laboratories Medical School University of Bristol 1 Biochemical aspects of mucus 2 Histochemical aspects of mucus 3 Conclusion References 1
Biochemical Aspects of Mncns
In the study of mucus a great many interests overlap, including those of clinicians, biochemists, histologists and physical chemists. This has resulted in a great deal of confusion in nomenclature. One of the earliest workers on mucus, Bostock (1805), castigated "the most esteemed medical and physiological writers" for using terms "in a vague and indeterminate manner" and the situation is no better nearly 200 years later. To take just one example, the term "mucoid" has been defined in at least four different ways: Clinicians have used "mucoid" to mean a non-purulent sputum (Miller, 1963; Medical Research Council, 1965). Biochemists have defined "mucoid" as any glycoprotein with a hexosamine content greater than 4% (Winder, 1958). Histochemists apply the word "mucoid" to any neutral "mucin" (Lev, 1970). Gynaecologists use "mucoid" for a type of cervical mucus (Moghissi & Neuhaus, 1966). Similar confusion exists for a host of other terms, such as "mucoprotein", "mucosubstance", "mucopolysaccharide", and so on. To overcome these problems of nomenclature biochemists have abandoned "vague and indeterminate" terms, particularly those prefixed by "muco-", together with elaborate and rigid classification systems such as those of Stacey (1946), Meyer (1953) and Winder (1958), and have replaced them with broad and rather general categories. "Glycoconjugate" is the term used to describe polymeric substances consisting of carbohydrate covalently linked to non-carbohydrate material, usually lipid or protein (nucleic acids are excluded from this nomenclature). There are two •Formerly of the Cardlothoradc Institute, Univcnity of London
VoL 34 No. 1
quite different types of glycoconjugate in which carbohydrate is linked to protein, namely the proteoglycans and the glycoproteins. The principal constituent of mucus and the component that gives the secretion its characteristic physicochemical properties is a glycoprotein of high molecular weight Some of the differences between proteoglycans and glycoproteins are given in Table I. Essentially a proteoglycan possesses long, unbranched carbohydrate chains (glycosaminoglycan), most of which have a repeating disaccharide structure. A typical glycosaminoglycan would therefore contain stretches consisting of hexuronic acid alternating with hexosamine. A glycoprotein, on the other hand, possesses relatively small carbohydrate units. The average size of these units is less than 10 monosaccharide residues and therefore, by common consent, they are all called oligosaccharide units, even though the range includes units that are considerably larger. The oligosaccharide units of glycoproteins are usually branched, have little or no repeating structure and do not contain hexuronic acid1. Glycoproteins comprise a wide range of materials with differing properties and functions. The purpose of Table II is to list the characteristics of mucus glycoproteins and to compare such characteristics with those of a quite different glycoprotein, namely a typical plasma globulin (virtually all plasma globulins are glycoproteins). In order to simplify the discussion, Tables I and n have both concentrated on the properties of mammalian, and particularly human, material. The linkage between carbohydrate and protein in mucus glycoproteins is termed "0-glycosidic" because the linkage is through an oxygen atom. The monosaccharide involved is JV-acetylgalactosamine, which is attached to either serine or threonine. In contrast, the linkage in plasma glycoproteins is termed 'W-glycosidic" because it is through a nitrogen atom from JV-acetylglucosamine to asparagine. The structure of these two types of linkage is shown by Morris & Rees (1978). The mucus glycoprotein, when freshly produced (native glycoprotein), has a molecular weight of several million, and well over half of this weight consists of carbohydrate. As the average size of the oligosaccharide units is only 8-10 monosaccharide residues, the glycoprotein is therefore an extended polypeptide chain with oligosaccharide units studded along its TABLE I. Differences between glycoproteins and proteoglycans Glycoprotein
Proteoglycan
Principal s i t u In the body
Membranes; body fluids and secretions (e.g. blood, mucus)
Carbohydrate component Size of carbohydrate component
Oligosaccharide unit
Skeletal and supporting tissues (e.g. cartilage, bone) Glycosaminoglycan More than 50 monosaccharIdes Xylose
Characteristic
Linkage monosaccharide Repeating structure
Less than 25 monosaccharldes N-Acetylhexosamine Little or none
Shape of carbohydrate Branched component Hexuronic add Absent
Repeating dlsaccharide Linear, unbranched Present
The above characteristics are typical of most glycoproteins and proteoglycans but, as always In biological substances, there aro exceptions. For example, keratin sulphate does not contain hexuronic add
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp TABLE II. Properties of mucus glycoproteins compared with those of plasma glycoproteins Characteristic
Mucus glycoproteln
Plasma glycoproteln
Amlno acid content
High levels of serine, threonineand proline. Low levels of aromatic and sulphur-containing imlno acids
Aminoacid spectrum of a typical protein
Carbohydrate content
More than 5 0 %
Less than 2 5 %
Linkage
N-Acetylgalactolamlne to serine or threonine (O-glycosidic)
N-Acetylglucosamlne to asparaglne (N-glycosIdlc)
Present
Present
Low levels or absent Present
Present Low levels or absent
Monosaccharides Fucose Galactose N-Acetylglucosamlne Slalicacld Mannose N-Aoetylgalactosamlne
There are several different types of glycoproteln but, for the purposes of this Table, mucus glycoproteins are compared with plasma glycoproteins. There are exceptions to the above statements: for example, lgA1, a plasma glycoprotein, contains O-glycosldically linked units
length attached to about every third amino acid. The oligosaccharide units project out from the polypeptide chain giving a structure that has been likened to a porcupine, a lamp brush or a test-tube brush, the acid groups (sialic acid and sulphate) being attached terminally to some of the bristles. The carbohydrate surrounds and shields the polypeptide core, for example, from the action of proteolytic enzymes. Although the above statements are true for the greater part of the polypeptide chain, there are carbohydrate-free stretches ("naked" peptide) which enable neighbouring chains to come together and be joined through the disulphide bonds of cystine. 2 Hlstocbemkal Aspects of Mucus There has also been confusion in the terms used to describe these substances in tissues. "Mucosubstances" is the one Pearse (1968) has adopted from the Spicer et al. (1965) classification. It seems timely to bring the histochemical description more into line with the biochemical, and we submit that to call the molecule "glycoprotein" while it is yet within the cell is probably not too presumptuous in our present stage of knowledge. Histochemistry combines histology with analytical chemistry, using carefully controlled techniques to identify and localize chemical substances in tissue on a cytological scale. One of the most effective ways of identifying the carbohydrate moiety is by the periodic acid/Schiff (PAS) method (McManus, 1946), which is based on satisfactory chemical methods and recognizes the presence of vicinal hydroxyl groups. A wide variety of stains has been developed to identify the various acid groups. They are of varying degrees of reliability but, as described later, a selection of them can be used to identify, even within a single cell, a range of acid residues.
The chemical uniformity is surprising in view of the variety to be seen in the staining of secretory granules within a single cell or even within a single granule. The relation between intracellular and extracellular features needs to be reconciled. Although the purified glycoprotein molecule, isolated by the chemist, contains both sulphate and sialic acid, these cannot necessarily be identified in all granules or even all cells, much less in a single granule (McCarthy & Reid, 1964; Jones & Reid, 1973). Does the isolated molecule represent crossUnking of smaller molecules that takes place outside the cell to produce a uniform structure? The biochemist can identify all the residues in the glycoprotein molecule. Within the tissues, the histochemist will need to be content with identifying some of the constituent residues, but it is only when these are synthesized into a glycoprotein molecule that they stain1. The polypeptide or sugar precursors do not give the characteristic staining during their passage through the endoplasmic reticulum or Golgi apparatus (Meyrick & Reid, 1975). It is only when they are packaged into the secretory granules that the characteristic staining is seen, and this staining is similar to that of the secretion after discharge when it is on the surface of the epithelium. Radioactive precursor can be traced through the cell organdies and recovered in the secretory granules and extracellular secretion (Sturgess & Reid, 1972a,b; Reid et al. 1976). For these reasons it seems justified to describe the secretory product within the cell in the same way as the extracellular product. If the terms are applied with some circumspection to the histochemical features then confusion should not arise and the histochemist's presumption is not too great. When no acid is identified, the product may be considered a neutral glycoprotein. This term does not preclude the presence of acid groups; it merely implies that they have not been identified histochemically. This material may represent the thinner secretions of the sol phase or may be a molecule with too little acid or acid not in a form to be detected histochemically. If this neutral molecule is cross-linked with other acid glycoproteins then its own true nature will not be apparent in the final secretion. It should be emphasized that, in those secretions shown by biochemical examination to contain little neutral glycoprotein, little of this substance can be identified histochemically at the site of origin, as is the case for airway secretion (Degand et al. 1973; Roberts, 1974). When sialic acid is identified, it is recommended that the terms "sialic-acid-containing glycoprotein"or "sialylated glycoprotein" be used; similarly, with sulphate, the terms "sulphatecontaining glycoprotein" or "sulphated glycoprotein" should be used. If both are found, "sialylated-sulphated glycoprotein" is appropriate rather than the terms "sulphomucin" or "sialomucin", since these terms suggest that only one acid radical is present. Sialylated and sulphated forms can be identified separately by histochemical methods, although such separate glycoproteins have not been isolated by the biochemist. This difference is another reason for suggesting that the product within the secreting granule may cross-link outside the cell to produce thefinalproduct. The nature of such aggregation is not clear, but purified glycoprotein has been found to aggregate spontaneously on standing (Creeth et al. 1977) while, in another system, its behaviour indicates that its molecules may spontaneously "disentangle" themselves with time (Sade
etal.1975). 1 See Jonei & Reid, pp. 9-16 of thta Bulletin.—ED.
Br. Med. Bull. 1978
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp 3
TABLE III. Some terms used in connection with mucus and related materials (Synonymous words or phnses are listed as "or . . . " . Words that have been used with different meanings have been listed and numbered according to frequency of use. The Italicized words have led to considerable confusion and it Is recommended that they are abandoned. The physicochemical terms were compiled by J M Creeth)
Ternu Blood-group glycoproteln; or blood-group substance; or blood-group antigen Bronchial mucus; or bronchial secretions Chaotrope; chaotroplc Debye distance Gel and sol
Glycoconjugate Glycopeptlde; glycopolypeptide Glycoproteln Glycosaminoglycan Heterosaccharlde; heteropolysaccharide Mucln Mucold
Mucopotytaccharlde Mucoprotcln Muatubttance
Mucus Mucus glycoprotein Ollgosaccharide unit; or sugar chains; or side chains; or carbohydrate moiety; or heterosaccharlde unit Polydlspenlty
Proteoglycan Sol Splnnbarkelt Sputum Visco-elastlclty
Viscosity
Vol. 34 No. 1
Definitions Mucus or mucus-type glycoproteln with certain bloodgroup activities Secretions from the respiratory tract below the larynx Literally, "dlsorder-lntrodudng". The term refers to those substances, chiefly salts, which perturb the structure of water, making it a denaturant medium A measure of the thickness of the ionic atmosphere around the charged groups in electrolyte solutions Often loosely used In the description of macro-molecular solutions. The sol is that part which Is In true solution, frequently of moderate to low viscosity. The gel, separable by low-speed centrlfugatlon, possesses a structure arising from intermolecular attractions, which leads to a degree of elasticity Carbohydrate covalently linked to non-carbohydrate material such as llpld or protein, but excluding nucleic acids Carbohydrate covalently linked to peptlde or polypeptlde. Usually obtained by degradation of glycoproteln Protein possessing covalently attached ollgosaccharide units (see Tables I and II) Carbohydrate moltty of proteoglycans (see Table I) Ollgosaccharide or polysaccharlde containing more than one type of monosaccharide I Mucus II Mucus glycoprotein (particularly In hlstochemlstry) I Non-purulent iputum II Glycoproteln iil Neutral mucln (hlstochemistry) iv Fraction of cervical mucus I Glycosaminoglycan il Mucus glycoproteln I Mucus glycoproteln II Proteoglycan i Vague term for any glycoproteln with a high carbohydrate content II General term Including mucus glycoprotelns and proteoglycans Total secretion from mucous membranes The principal glycoproteln component of mucus (see Table II) The Individual carbohydrate unit of glycoprotelns
Used mostly to describe the spread of molecular weights in polymers. A macromolecular preparation may be "homogeneous" (containing no other kind of molecule) but be either "monodlsperse" (as with a pure protein), or "polydlsperse" if it contains molecules of varying degrees of polymerization. Polysaccharides and mucus glycoprotelns are examples of materials- that are polydlsperse Carbohydrate (glycosaminoglycan) covalently attached to protein (see Table I) See Gel and sol above Term used principally In connectlon.wlth cervical mucus, to describe the ability of mucus to be drawn Into threads Expectorated secretions from the respiratory tract together with saliva The property exhibited by gels of partial recovery from shear stress. This property is between that of a completely elastic solid, which shows complete recovery from stress, and a purely viscous liquid, which exhibits no recovery A measure of the resistance to flow, specifically the ratio of shear stress to rate of shear "strain. A Newtonian liquid has a constant viscosity, Independent of shear stress; mucus Is non-Newtonian and the viscosity decreases markedly with Increasing stress. (Intrinsic viscosity refers to a macromolecular solute and is a measure of the effective volume of the molecule)
Conclusion
We suggest that "mucus" be used to describe the slimy secretion of the various epithelial surfaces being considered in this Bulletin and that, as far as possible, this word be used both as noun and adjective where other substances or features call for description. The purified glycoprotein from mucus can then be called "mucus glycoprotein". This in in contrast to plasma glycoproteins, which are present in many of the epithelial secretions. There are probably two occasions when it will be preferable to use the word "mucous", namely in mucous membrane and mucous cell, to pair with the corresponding serous type. Sputum may be purulent or non-purulent. Since both purulent and non-purulent sputa contain mucus, non-purulent is probably a better description of uninfected sputum than mucoid, although long usage has engrained the use of that word. It should be possible to drop various words prefixed by "muco-" that have been used to describe macroscopic appearance or chemical structure. And so the mucosa, of airway or gut for example, secretes mucus from which can be isolated and purified the mucus glycoprotein. This is mixed with other macromolecules, such as plasma glycoproteins, and with smaller molecules to make up the total secretion. In the case of the airways, sputum by convention describes the material that is expectorated. It is not applied to bronchial secretion until it is above the larynx. The expectorated product is mixed also with saliva and perhaps with nasal secretion. Taking this convention into account, the mixture does not present a semantic problem, although it may be a problem for the clinician. Centrifugation separates the sputum into two layers—the sol and the gel—in both of which are mucus glycoproteins. In a similar way sputum, on standing, separates into two layers. The mucus recovered from the airways has the properties of a visco-elastic gel. Since it is a non-Newtonian substance, viscosity varies with the shear rate applied. It is "apparent viscosity" that is measured, and so its value is qualified by the shear rate at which the measurement is made. "Stickiness" and "pourability" are also obvious properties of the secretion, and simple and reproducible ways of measuring these properties have been developed. To those readers interested in more details about the physical properties of the mucus secretions, the monograph An introduction to biorheology by Scott Blair (1974) can be recommended, either as an introduction to the subject or for reference to more advanced discussion. Finally, the various terms used in the description of these substances are summarized in Table HI.
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp REFERENCES
Blair G W Scott (1974) An introduction to blorheology. Elsevier Amsterdam Bostock J (1805) / . Nat. Philos. Chem, Arts, 11, 244-254 Creeth J M, Bhaskar K R, Horton J R, Das I, Lopez-Vidriero M T & Reid L (1977) Biochem. J. 167, 557-569 Degand P, Roussel P, Lamblin G & Havez R (1973) Biochim. Biophys. Acta, 320, 318-330 Jones R & Reid L (1973) Histochem. J. 5,9-18,19-27 Lev R (1970) Prog. Gastroenterol. 2,13-41 McCarthy C & Reid L (1964) Q. J. Exp. Pathol. 49, 85-94 McManus J F A (1946) Nature {London) 158, 202 [Letter] Medical Research Council: Committee on the Aetiology of Chronic Bronchitis (1965) Lancet, 1, 775-779 Meyer K (1953) In: Cole W H , ed. Some conjugated proteins, pp. 64-73 (Proceedings of the Ninth Annual Conference on Protein Metabolism, 30-31 January 1953). Rutgers University Press, New Brunswick, N.J. Meyrick B & Reid L (1975) / . Cell Biol. 67, 320-344 Miller D L (1963) Am. Rev. Respir. Dis. 88, 473-483
Moghissi K S & Neuhaus O W (1966) Am.J. Obstet. Gynecol. 96,91-95 Morris E R & Rees D A (1978) Br. Med. Bull. 34,49-53 Pearse A G E (1968) Histochemistry: theoretical and applied, 3rd ed., vol. 1. Churchill, London ReidL, Meyrick B & ColesS (1976) In: Balls M & Monnickendam M A, ed. Organ culture in biomedical research, pp. 463-480 (British Society for Cell Biology symposium 1). Cambridge University Press, London Roberts G P (1974) Eur. J. Biochem. 50, 265-280 Sad6 J, Meyer F A, King M & Silberberg A (1975) Acta Otolaryngol. 79,277-282 Spicer S S, Leppi T J & Stoward P J (1965) / . Histochem. Cytoehem. 13, 599-603 Stacey M (1946) Adv. Carbohydr. Chem. 2, 161-201 Sturgess J & Reid L (1972a) Clin. Sci. 43, 533-543 Sturgess J & Reid (1972b) Exp. Mol. Pathol. 16, 362-381 Winder R J (1958) In: Wolstenholme G E W & O'Connor M, ed. Ciba Foundation symposium on the chemistry and biology of mucopotysaccharldes, pp. 245-263. Churchill, London
Br. Med. BuIL 1978
PLATE I
SECRETORY CELLS AND THEIR GLYCOPROTEINS Rosemary Jones & Lynne Reid (FIG. A-E)
A. Mucous cells of the human bronchial submucosal gland showing confluent electron-lucent granules (mg) Meyrick (1977) by permission of Plenum Press
B. Serous cells of the human bronchial submucosal gland showing discrete electron-dense granules (sg) Meyrlck & Reid (1975) by permission of the Journal of Cell Biology (Both fifuret ire electron micrographs, prepared uiinf glutaraldehyde tnd osmium tetroxide/uranyl acatate and laad citrate; magnification x 5100)
PLATE II
SECRETORY CELLS AND THEIR GLYCOPROTEINS (continued) C. The range of cell types in the human tracheobronchlal submucosal gland. In bars i-iv of the histogram (which have areas In black, as well as cross-hatched, hatched and unfilled areas), the density represents the degree of acidity demonstrated by each technique. In bar v, the various sizes of dots denote the degree of uptake from most dense -*• no uptake.
After neuraminidase treatment
i: the total area of acid glycoprotein in the gland; II and Ml: the removal of sialic acid by (ii) the enzyme neuraminidase and (III) acid hydrolysis; Iv: the area staining with special sulphate stains, I.e., aldehyde fuchsin/Alcian Blue, high iron diamine/Alclan Blue; v: the concentration of uptake of sodium [3SS]sulphate from organ culture, in regions of the gland that correspond to those In the bars above.
After acid hydrolysis Stains for sulphated glycoprotein Sodium ["S]sulphate uptake
From knowledge of the proportion of gland that includes one type of acid glycoprotein the proportion occupied by the others can be predicted
i Rats exposed to
tobacco smoke for: 6 weeks 2 weeks
100 -i
D. Tracheal secretory cells of each type in control rats and rats exposed to tobacco smoke for two or six weeks. For an explanation of this diagram see section 5 of the text
KEY: I I tS^i aBI t'ysM
7.0
10
20
30
40
5O
60
70
80
90
large numbtr of granules stained with PAS small number of granules stained with PAS larfa number of granules stained with AB or AB/PAS small number of granules stained with AB or AB/PAS
100
Control pigs |
%0
10
20
30
40
34%
50
11%]
60
Pigs with enzootic pneumonia I 67,1
tSm^j neutral glycoprotein |
| acid glycoprotein
70
80
90
E. Area of neutral and acid glycoprotein staining and the proportion of the latter Including each acid type, mucous cells of bronchial submucosal gland in control pigs and In plgj with enzootic pneumonia
100
KEY: A: slalylated glycoprotein sensitive to neuraminidase B: slalylated glycoprotein resistant to neuraminidase C: sulphated glycoprotein
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp
THE BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS LYNNE REED* MD FRCP FRCPath Harvard Medical School Boston, Massachusetts, USA and The Children's Hospital Medical Center Boston, Massachusetts, USA
J R CLAMP MD PhD FRIC MRCP Clinical Research Laboratories Medical School University of Bristol 1 Biochemical aspects of mucus 2 Histochemical aspects of mucus 3 Conclusion References 1
Biochemical Aspects of Mncns
In the study of mucus a great many interests overlap, including those of clinicians, biochemists, histologists and physical chemists. This has resulted in a great deal of confusion in nomenclature. One of the earliest workers on mucus, Bostock (1805), castigated "the most esteemed medical and physiological writers" for using terms "in a vague and indeterminate manner" and the situation is no better nearly 200 years later. To take just one example, the term "mucoid" has been defined in at least four different ways: Clinicians have used "mucoid" to mean a non-purulent sputum (Miller, 1963; Medical Research Council, 1965). Biochemists have defined "mucoid" as any glycoprotein with a hexosamine content greater than 4% (Winder, 1958). Histochemists apply the word "mucoid" to any neutral "mucin" (Lev, 1970). Gynaecologists use "mucoid" for a type of cervical mucus (Moghissi & Neuhaus, 1966). Similar confusion exists for a host of other terms, such as "mucoprotein", "mucosubstance", "mucopolysaccharide", and so on. To overcome these problems of nomenclature biochemists have abandoned "vague and indeterminate" terms, particularly those prefixed by "muco-", together with elaborate and rigid classification systems such as those of Stacey (1946), Meyer (1953) and Winder (1958), and have replaced them with broad and rather general categories. "Glycoconjugate" is the term used to describe polymeric substances consisting of carbohydrate covalently linked to non-carbohydrate material, usually lipid or protein (nucleic acids are excluded from this nomenclature). There are two •Formerly of the Cardlothoradc Institute, Univcnity of London
VoL 34 No. 1
quite different types of glycoconjugate in which carbohydrate is linked to protein, namely the proteoglycans and the glycoproteins. The principal constituent of mucus and the component that gives the secretion its characteristic physicochemical properties is a glycoprotein of high molecular weight Some of the differences between proteoglycans and glycoproteins are given in Table I. Essentially a proteoglycan possesses long, unbranched carbohydrate chains (glycosaminoglycan), most of which have a repeating disaccharide structure. A typical glycosaminoglycan would therefore contain stretches consisting of hexuronic acid alternating with hexosamine. A glycoprotein, on the other hand, possesses relatively small carbohydrate units. The average size of these units is less than 10 monosaccharide residues and therefore, by common consent, they are all called oligosaccharide units, even though the range includes units that are considerably larger. The oligosaccharide units of glycoproteins are usually branched, have little or no repeating structure and do not contain hexuronic acid1. Glycoproteins comprise a wide range of materials with differing properties and functions. The purpose of Table II is to list the characteristics of mucus glycoproteins and to compare such characteristics with those of a quite different glycoprotein, namely a typical plasma globulin (virtually all plasma globulins are glycoproteins). In order to simplify the discussion, Tables I and n have both concentrated on the properties of mammalian, and particularly human, material. The linkage between carbohydrate and protein in mucus glycoproteins is termed "0-glycosidic" because the linkage is through an oxygen atom. The monosaccharide involved is JV-acetylgalactosamine, which is attached to either serine or threonine. In contrast, the linkage in plasma glycoproteins is termed 'W-glycosidic" because it is through a nitrogen atom from JV-acetylglucosamine to asparagine. The structure of these two types of linkage is shown by Morris & Rees (1978). The mucus glycoprotein, when freshly produced (native glycoprotein), has a molecular weight of several million, and well over half of this weight consists of carbohydrate. As the average size of the oligosaccharide units is only 8-10 monosaccharide residues, the glycoprotein is therefore an extended polypeptide chain with oligosaccharide units studded along its TABLE I. Differences between glycoproteins and proteoglycans Glycoprotein
Proteoglycan
Principal s i t u In the body
Membranes; body fluids and secretions (e.g. blood, mucus)
Carbohydrate component Size of carbohydrate component
Oligosaccharide unit
Skeletal and supporting tissues (e.g. cartilage, bone) Glycosaminoglycan More than 50 monosaccharIdes Xylose
Characteristic
Linkage monosaccharide Repeating structure
Less than 25 monosaccharldes N-Acetylhexosamine Little or none
Shape of carbohydrate Branched component Hexuronic add Absent
Repeating dlsaccharide Linear, unbranched Present
The above characteristics are typical of most glycoproteins and proteoglycans but, as always In biological substances, there aro exceptions. For example, keratin sulphate does not contain hexuronic add
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp TABLE II. Properties of mucus glycoproteins compared with those of plasma glycoproteins Characteristic
Mucus glycoproteln
Plasma glycoproteln
Amlno acid content
High levels of serine, threonineand proline. Low levels of aromatic and sulphur-containing imlno acids
Aminoacid spectrum of a typical protein
Carbohydrate content
More than 5 0 %
Less than 2 5 %
Linkage
N-Acetylgalactolamlne to serine or threonine (O-glycosidic)
N-Acetylglucosamlne to asparaglne (N-glycosIdlc)
Present
Present
Low levels or absent Present
Present Low levels or absent
Monosaccharides Fucose Galactose N-Acetylglucosamlne Slalicacld Mannose N-Aoetylgalactosamlne
There are several different types of glycoproteln but, for the purposes of this Table, mucus glycoproteins are compared with plasma glycoproteins. There are exceptions to the above statements: for example, lgA1, a plasma glycoprotein, contains O-glycosldically linked units
length attached to about every third amino acid. The oligosaccharide units project out from the polypeptide chain giving a structure that has been likened to a porcupine, a lamp brush or a test-tube brush, the acid groups (sialic acid and sulphate) being attached terminally to some of the bristles. The carbohydrate surrounds and shields the polypeptide core, for example, from the action of proteolytic enzymes. Although the above statements are true for the greater part of the polypeptide chain, there are carbohydrate-free stretches ("naked" peptide) which enable neighbouring chains to come together and be joined through the disulphide bonds of cystine. 2 Hlstocbemkal Aspects of Mucus There has also been confusion in the terms used to describe these substances in tissues. "Mucosubstances" is the one Pearse (1968) has adopted from the Spicer et al. (1965) classification. It seems timely to bring the histochemical description more into line with the biochemical, and we submit that to call the molecule "glycoprotein" while it is yet within the cell is probably not too presumptuous in our present stage of knowledge. Histochemistry combines histology with analytical chemistry, using carefully controlled techniques to identify and localize chemical substances in tissue on a cytological scale. One of the most effective ways of identifying the carbohydrate moiety is by the periodic acid/Schiff (PAS) method (McManus, 1946), which is based on satisfactory chemical methods and recognizes the presence of vicinal hydroxyl groups. A wide variety of stains has been developed to identify the various acid groups. They are of varying degrees of reliability but, as described later, a selection of them can be used to identify, even within a single cell, a range of acid residues.
The chemical uniformity is surprising in view of the variety to be seen in the staining of secretory granules within a single cell or even within a single granule. The relation between intracellular and extracellular features needs to be reconciled. Although the purified glycoprotein molecule, isolated by the chemist, contains both sulphate and sialic acid, these cannot necessarily be identified in all granules or even all cells, much less in a single granule (McCarthy & Reid, 1964; Jones & Reid, 1973). Does the isolated molecule represent crossUnking of smaller molecules that takes place outside the cell to produce a uniform structure? The biochemist can identify all the residues in the glycoprotein molecule. Within the tissues, the histochemist will need to be content with identifying some of the constituent residues, but it is only when these are synthesized into a glycoprotein molecule that they stain1. The polypeptide or sugar precursors do not give the characteristic staining during their passage through the endoplasmic reticulum or Golgi apparatus (Meyrick & Reid, 1975). It is only when they are packaged into the secretory granules that the characteristic staining is seen, and this staining is similar to that of the secretion after discharge when it is on the surface of the epithelium. Radioactive precursor can be traced through the cell organdies and recovered in the secretory granules and extracellular secretion (Sturgess & Reid, 1972a,b; Reid et al. 1976). For these reasons it seems justified to describe the secretory product within the cell in the same way as the extracellular product. If the terms are applied with some circumspection to the histochemical features then confusion should not arise and the histochemist's presumption is not too great. When no acid is identified, the product may be considered a neutral glycoprotein. This term does not preclude the presence of acid groups; it merely implies that they have not been identified histochemically. This material may represent the thinner secretions of the sol phase or may be a molecule with too little acid or acid not in a form to be detected histochemically. If this neutral molecule is cross-linked with other acid glycoproteins then its own true nature will not be apparent in the final secretion. It should be emphasized that, in those secretions shown by biochemical examination to contain little neutral glycoprotein, little of this substance can be identified histochemically at the site of origin, as is the case for airway secretion (Degand et al. 1973; Roberts, 1974). When sialic acid is identified, it is recommended that the terms "sialic-acid-containing glycoprotein"or "sialylated glycoprotein" be used; similarly, with sulphate, the terms "sulphatecontaining glycoprotein" or "sulphated glycoprotein" should be used. If both are found, "sialylated-sulphated glycoprotein" is appropriate rather than the terms "sulphomucin" or "sialomucin", since these terms suggest that only one acid radical is present. Sialylated and sulphated forms can be identified separately by histochemical methods, although such separate glycoproteins have not been isolated by the biochemist. This difference is another reason for suggesting that the product within the secreting granule may cross-link outside the cell to produce thefinalproduct. The nature of such aggregation is not clear, but purified glycoprotein has been found to aggregate spontaneously on standing (Creeth et al. 1977) while, in another system, its behaviour indicates that its molecules may spontaneously "disentangle" themselves with time (Sade
etal.1975). 1 See Jonei & Reid, pp. 9-16 of thta Bulletin.—ED.
Br. Med. Bull. 1978
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp 3
TABLE III. Some terms used in connection with mucus and related materials (Synonymous words or phnses are listed as "or . . . " . Words that have been used with different meanings have been listed and numbered according to frequency of use. The Italicized words have led to considerable confusion and it Is recommended that they are abandoned. The physicochemical terms were compiled by J M Creeth)
Ternu Blood-group glycoproteln; or blood-group substance; or blood-group antigen Bronchial mucus; or bronchial secretions Chaotrope; chaotroplc Debye distance Gel and sol
Glycoconjugate Glycopeptlde; glycopolypeptide Glycoproteln Glycosaminoglycan Heterosaccharlde; heteropolysaccharide Mucln Mucold
Mucopotytaccharlde Mucoprotcln Muatubttance
Mucus Mucus glycoprotein Ollgosaccharide unit; or sugar chains; or side chains; or carbohydrate moiety; or heterosaccharlde unit Polydlspenlty
Proteoglycan Sol Splnnbarkelt Sputum Visco-elastlclty
Viscosity
Vol. 34 No. 1
Definitions Mucus or mucus-type glycoproteln with certain bloodgroup activities Secretions from the respiratory tract below the larynx Literally, "dlsorder-lntrodudng". The term refers to those substances, chiefly salts, which perturb the structure of water, making it a denaturant medium A measure of the thickness of the ionic atmosphere around the charged groups in electrolyte solutions Often loosely used In the description of macro-molecular solutions. The sol is that part which Is In true solution, frequently of moderate to low viscosity. The gel, separable by low-speed centrlfugatlon, possesses a structure arising from intermolecular attractions, which leads to a degree of elasticity Carbohydrate covalently linked to non-carbohydrate material such as llpld or protein, but excluding nucleic acids Carbohydrate covalently linked to peptlde or polypeptlde. Usually obtained by degradation of glycoproteln Protein possessing covalently attached ollgosaccharide units (see Tables I and II) Carbohydrate moltty of proteoglycans (see Table I) Ollgosaccharide or polysaccharlde containing more than one type of monosaccharide I Mucus II Mucus glycoprotein (particularly In hlstochemlstry) I Non-purulent iputum II Glycoproteln iil Neutral mucln (hlstochemistry) iv Fraction of cervical mucus I Glycosaminoglycan il Mucus glycoproteln I Mucus glycoproteln II Proteoglycan i Vague term for any glycoproteln with a high carbohydrate content II General term Including mucus glycoprotelns and proteoglycans Total secretion from mucous membranes The principal glycoproteln component of mucus (see Table II) The Individual carbohydrate unit of glycoprotelns
Used mostly to describe the spread of molecular weights in polymers. A macromolecular preparation may be "homogeneous" (containing no other kind of molecule) but be either "monodlsperse" (as with a pure protein), or "polydlsperse" if it contains molecules of varying degrees of polymerization. Polysaccharides and mucus glycoprotelns are examples of materials- that are polydlsperse Carbohydrate (glycosaminoglycan) covalently attached to protein (see Table I) See Gel and sol above Term used principally In connectlon.wlth cervical mucus, to describe the ability of mucus to be drawn Into threads Expectorated secretions from the respiratory tract together with saliva The property exhibited by gels of partial recovery from shear stress. This property is between that of a completely elastic solid, which shows complete recovery from stress, and a purely viscous liquid, which exhibits no recovery A measure of the resistance to flow, specifically the ratio of shear stress to rate of shear "strain. A Newtonian liquid has a constant viscosity, Independent of shear stress; mucus Is non-Newtonian and the viscosity decreases markedly with Increasing stress. (Intrinsic viscosity refers to a macromolecular solute and is a measure of the effective volume of the molecule)
Conclusion
We suggest that "mucus" be used to describe the slimy secretion of the various epithelial surfaces being considered in this Bulletin and that, as far as possible, this word be used both as noun and adjective where other substances or features call for description. The purified glycoprotein from mucus can then be called "mucus glycoprotein". This in in contrast to plasma glycoproteins, which are present in many of the epithelial secretions. There are probably two occasions when it will be preferable to use the word "mucous", namely in mucous membrane and mucous cell, to pair with the corresponding serous type. Sputum may be purulent or non-purulent. Since both purulent and non-purulent sputa contain mucus, non-purulent is probably a better description of uninfected sputum than mucoid, although long usage has engrained the use of that word. It should be possible to drop various words prefixed by "muco-" that have been used to describe macroscopic appearance or chemical structure. And so the mucosa, of airway or gut for example, secretes mucus from which can be isolated and purified the mucus glycoprotein. This is mixed with other macromolecules, such as plasma glycoproteins, and with smaller molecules to make up the total secretion. In the case of the airways, sputum by convention describes the material that is expectorated. It is not applied to bronchial secretion until it is above the larynx. The expectorated product is mixed also with saliva and perhaps with nasal secretion. Taking this convention into account, the mixture does not present a semantic problem, although it may be a problem for the clinician. Centrifugation separates the sputum into two layers—the sol and the gel—in both of which are mucus glycoproteins. In a similar way sputum, on standing, separates into two layers. The mucus recovered from the airways has the properties of a visco-elastic gel. Since it is a non-Newtonian substance, viscosity varies with the shear rate applied. It is "apparent viscosity" that is measured, and so its value is qualified by the shear rate at which the measurement is made. "Stickiness" and "pourability" are also obvious properties of the secretion, and simple and reproducible ways of measuring these properties have been developed. To those readers interested in more details about the physical properties of the mucus secretions, the monograph An introduction to biorheology by Scott Blair (1974) can be recommended, either as an introduction to the subject or for reference to more advanced discussion. Finally, the various terms used in the description of these substances are summarized in Table HI.
BIOCHEMICAL AND HISTOCHEMICAL NOMENCLATURE OF MUCUS L Reid & J R Clamp REFERENCES
Blair G W Scott (1974) An introduction to blorheology. Elsevier Amsterdam Bostock J (1805) / . Nat. Philos. Chem, Arts, 11, 244-254 Creeth J M, Bhaskar K R, Horton J R, Das I, Lopez-Vidriero M T & Reid L (1977) Biochem. J. 167, 557-569 Degand P, Roussel P, Lamblin G & Havez R (1973) Biochim. Biophys. Acta, 320, 318-330 Jones R & Reid L (1973) Histochem. J. 5,9-18,19-27 Lev R (1970) Prog. Gastroenterol. 2,13-41 McCarthy C & Reid L (1964) Q. J. Exp. Pathol. 49, 85-94 McManus J F A (1946) Nature {London) 158, 202 [Letter] Medical Research Council: Committee on the Aetiology of Chronic Bronchitis (1965) Lancet, 1, 775-779 Meyer K (1953) In: Cole W H , ed. Some conjugated proteins, pp. 64-73 (Proceedings of the Ninth Annual Conference on Protein Metabolism, 30-31 January 1953). Rutgers University Press, New Brunswick, N.J. Meyrick B & Reid L (1975) / . Cell Biol. 67, 320-344 Miller D L (1963) Am. Rev. Respir. Dis. 88, 473-483
Moghissi K S & Neuhaus O W (1966) Am.J. Obstet. Gynecol. 96,91-95 Morris E R & Rees D A (1978) Br. Med. Bull. 34,49-53 Pearse A G E (1968) Histochemistry: theoretical and applied, 3rd ed., vol. 1. Churchill, London ReidL, Meyrick B & ColesS (1976) In: Balls M & Monnickendam M A, ed. Organ culture in biomedical research, pp. 463-480 (British Society for Cell Biology symposium 1). Cambridge University Press, London Roberts G P (1974) Eur. J. Biochem. 50, 265-280 Sad6 J, Meyer F A, King M & Silberberg A (1975) Acta Otolaryngol. 79,277-282 Spicer S S, Leppi T J & Stoward P J (1965) / . Histochem. Cytoehem. 13, 599-603 Stacey M (1946) Adv. Carbohydr. Chem. 2, 161-201 Sturgess J & Reid L (1972a) Clin. Sci. 43, 533-543 Sturgess J & Reid (1972b) Exp. Mol. Pathol. 16, 362-381 Winder R J (1958) In: Wolstenholme G E W & O'Connor M, ed. Ciba Foundation symposium on the chemistry and biology of mucopotysaccharldes, pp. 245-263. Churchill, London
Br. Med. BuIL 1978
PLATE I
SECRETORY CELLS AND THEIR GLYCOPROTEINS Rosemary Jones & Lynne Reid (FIG. A-E)
A. Mucous cells of the human bronchial submucosal gland showing confluent electron-lucent granules (mg) Meyrick (1977) by permission of Plenum Press
B. Serous cells of the human bronchial submucosal gland showing discrete electron-dense granules (sg) Meyrlck & Reid (1975) by permission of the Journal of Cell Biology (Both fifuret ire electron micrographs, prepared uiinf glutaraldehyde tnd osmium tetroxide/uranyl acatate and laad citrate; magnification x 5100)
PLATE II
SECRETORY CELLS AND THEIR GLYCOPROTEINS (continued) C. The range of cell types in the human tracheobronchlal submucosal gland. In bars i-iv of the histogram (which have areas In black, as well as cross-hatched, hatched and unfilled areas), the density represents the degree of acidity demonstrated by each technique. In bar v, the various sizes of dots denote the degree of uptake from most dense -*• no uptake.
After neuraminidase treatment
i: the total area of acid glycoprotein in the gland; II and Ml: the removal of sialic acid by (ii) the enzyme neuraminidase and (III) acid hydrolysis; Iv: the area staining with special sulphate stains, I.e., aldehyde fuchsin/Alcian Blue, high iron diamine/Alclan Blue; v: the concentration of uptake of sodium [3SS]sulphate from organ culture, in regions of the gland that correspond to those In the bars above.
After acid hydrolysis Stains for sulphated glycoprotein Sodium ["S]sulphate uptake
From knowledge of the proportion of gland that includes one type of acid glycoprotein the proportion occupied by the others can be predicted
i Rats exposed to
tobacco smoke for: 6 weeks 2 weeks
100 -i
D. Tracheal secretory cells of each type in control rats and rats exposed to tobacco smoke for two or six weeks. For an explanation of this diagram see section 5 of the text
KEY: I I tS^i aBI t'ysM
7.0
10
20
30
40
5O
60
70
80
90
large numbtr of granules stained with PAS small number of granules stained with PAS larfa number of granules stained with AB or AB/PAS small number of granules stained with AB or AB/PAS
100
Control pigs |
%0
10
20
30
40
34%
50
11%]
60
Pigs with enzootic pneumonia I 67,1
tSm^j neutral glycoprotein |
| acid glycoprotein
70
80
90
E. Area of neutral and acid glycoprotein staining and the proportion of the latter Including each acid type, mucous cells of bronchial submucosal gland in control pigs and In plgj with enzootic pneumonia
100
KEY: A: slalylated glycoprotein sensitive to neuraminidase B: slalylated glycoprotein resistant to neuraminidase C: sulphated glycoprotein
