John R. Clamp

PHYLOGENETIC ASPECTS OF C-REACTIVE PROTEIN AND RELATED PROTEINS*

C-reactive protein (CRP) was discovered by Tillett and Francis’ in the sera of patients with various infectious and inflammatory diseases as a material which precipitated pneumococcal C-polysaccharide (CPS). Subsequently Abernethy and Ave

The roles of enteric bacterial sialidase, sialate O-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

characterized CRP as a protein and identified the requirement for calcium ions in its interaction with CPS, while they and established that the appearance of CRP in the serum is a nonspecific response to infection, inflammation and tissue damage. Abernethy and Averf also introduced the term “acute phase sera” to designate samples obtained from patients in the acute phase of infectious diseases. CRP was called the “acute phase protein” and this term was subsequently applied to the large number of other plasma proteins, the concentrations of which are raised in acute phase sera. At an early stage Abernethy’ reported the presence of a precipitin comparable to CRP in acute phase monkey serum and, although he had been unable to find any in mouse or rabbit sera, Anderson and McCarty’later described the existence

C-reactive protein and serum amyloid P component in the plaice (Pleuronectes platessa L.), a marine teleost, are homologous with their human counterparts

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or moreO-acetyl esters at positions C7–C9 on the sialic acids retarded the rate of hydrolysis. A specific sialateO-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and aKM of approximately 1mm sialateO-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[3H]ol 6-O-sulfate with a pH optimum of pH 5.0 and aKM of approximately 1mm.Faecal extracts from ulcerative colitis (UC) patients had higher sialateO-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged.Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acidO-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects.

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland

C-reactive protein and serum amyloid P component were isolated from serum of the plaice (Pleuronectes platessa L.), a murine teleost. The isolation was based on their calcium-dependent binding affinity for pneumococcal C-polysaccharide and for agarose, respectively. These specificities are the same as those of human C-reactive protein and serum amyloid P component, respectively, and we have previously reported that the plaice molecules resemble human C-reactive protein and serum amyloid P component in their electron microscopic appearance. We describe here estimation of the molecular weights of plaice C-reactive protein and serum amyloid P component and their subunits, and analysis of their amino acid composition, glycosylation and partial amino-terminal amino acid sequences. The results establish that plaice C-reactive protein and serum amyloid P component are homologous with each other and with their human counterparts and indicate that there has been stable conservation of this protein family throughout vertebrate evolution.

Structural analysis of the carbohydrate moieties of glycoproteins by regiospecific degradation and liquid chromatography

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×105 Da and aggregates in excess of 106 Da, while the minor mucin ranged from 3.0 × 105 to 106 Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.

A comparison of the results of sequential hydrazinolysis-nitrosation and alkali-mediated cleavage-nitrosation of theO-linked oligosaccharides of gastric mucus glycoproteins

Abstract The reaction sequence of hydrazinolysis, nitrosation, and reduction, followed by liquid chromatography (l.c.) has been studied as a method for the routine structural analysis of the asparagine-bound oligosaccharides of glycoproteins. Glycopeptides derived from IgM and ovalbumin by proteolysis were used as test materials. The hydrazinium sulphate-catalysed hydrazinolysis was superior to the longer uncatalysed reaction, in that there was less non-specific degradation and higher degree of N -deacetylation. The nitrosation products were reduced in situ with sodium cyanoborohydride, and the l.c. analysis required 20 min for the fractionation of oligosaccharides up to decasaccharide. The l.c. profile is characteristic of the structure of the carbohydrate unit. The analytical l.c. column may also be used to isolate oligosaccharide fractions in quantities of several hundred micrograms.

The glycoconjugate content of human faeces and ileostomy effluents

Analysis of the oligosaccharides released from pig gastric mucus glycopolypeptides by hydrazinolysis showed that degradation had occurred. Nitrosation of the products followed by reduction gave a mixture that had a low content of 2,5-anhydro-D-talitol, which implied destruction of much of the terminal reducing 2-amino-2-deoxy-D-galactose. Under the conditions of hydrazinolysis, cellobiose was largely unchanged but laminaribiose gave a complex mixture that probably contained glucose hydrazone (13C-n.m.r. data). In order to avoid degradation, the hydrazinolysis-nitrosation sequence should be applied to the reduced oligosaccharides released on cleavage with alkali.

Some aspects of the glycoprotein and glycopolypeptide content of human gastric mucus

Normal human faeces and effluent from terminal ileostomies in patients with ulcerative colitis were collected. The results for ileostomy effluent given as g/24 h (mean +/- SD; n = 12) were as follows: wet weight, 897 +/- 120; total dry weight, 84 +/- 8; non-diffusible dry weight 46 +/- 5. The corresponding results for faeces (n = 6) were 104 +/- 24; 26 +/- 6; 19 +/- 5. Whereas approximately 15% of the total dry weight of ileostomy effluent appeared to consist of mucin-derived material, only trace amounts of such material could be detected in faeces.

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours

The carbohydrate content of non-diffusable, glycoprotein and glycopolypeptide material has been studied in normal human gastric aspirates. Pentagastrin doubles the volume of secretions but has no effect on the amount of non-diffusable material. Only about 40% of the weight of the non-diffusable material is mucin in nature. Gel-permeation chromatography indicated that about half of the mucin survived as high molecular mass glycoprotein. Monosaccharide differences, for example between secretors and non-secretors, only became manifest at the glycopolypeptide stage. These results emphasize the dangers of attempting to assess mucin changes by simple carbohydrate analyses of unfractionated gastric aspirates.

Studies Upon the Secretion of Gastric Mucus from Normal Subjects

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.