Human respiratory tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial secretions,and sputum of patients with bronchitis and cystic fibrosis

Mucous glycoproteins were isolated by agarose gel filtration from nonpurulent tracheobronchial secretions and purulent sputum which had been reduced, carboxymethylated and, in the case of purulent secretions, treated with deoxyribonuclease. The solubilized and purified glycoproteins were fractionated on diethylaminoethyl cellulose into two major (I, II) and two minor (Ia, III) blood group active components. Components I and II had similar carbohydrate and amino acid compositions which were typical for human blood group substances. These two components did differ in several respects. Component I contained 1.4–2.6% sulfate and did not inhibit influenza virus hemagglutination while component II contained 7.1–7.8% sulfate and was a potent inhibitor of virus hemagglutination. Component II also migrated more rapidly on sodium dodecyl sulfate-3.3% acrylamide gel electrophoresis. Components I and II in purulent secretions displayed only minor compositional differences from their counterparts in nonpurulent secretions. Component II was more abundant in two sputum samples from subjects with cystic fibrosis than in purulent bronchitic secretions or in nonpurulent secretions.     

Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces "mucus" with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.     

Isolation and characterization of glycoproteins from canine tracheal pouch secretions.

Canine tracheal pouch secretions were solubilized with 1% sodium dodecyl sulfate and visualized by sodium dodecyl sulfate-agarose-acrylamide gel electrophoresis. Intact mucus, and water-soluble and insoluble fractions of mucus were shown to be composed of high molecular weight glycoproteins (Mr greater than or equal to 3 . 10(6)) and three major classes of proteins of lower molecular weight (Mr approximately 4 . 10(5), 2 . 10(5), and 6 . 10(4)). When the mucus secretions were further treated with a reducing agent, the glycoproteins were dissociated into subunits which appeared on the gel as three discrete bands. Separation of the high molecular weight glycoproteins from the other proteins was achieved by gel filtration on Biogel A-15m in the presence of 1% dodecyl sulfate following reduction and alkylation of mucus. These glycoproteins were further resolved, using DEAE cellulose chromatography in the presence of 6 M urea, into two protein fractions. Both fractions contained approximately 87% carbohydrate, high amounts of serine and threonine but differed significantly in contents of N-acetyl glucosamine and sialic acid; their mobility on gel electrophoresis was also different. Significant contents of cysteine were noted in both fractions. Results of this study indicate that the canine tracheal pouch preparations provide normal tracheal secretions which bear similarity in structure to the tracheobronchial secretions obtained from human patients.     

Analysis of the conformation and stability of human bronchial lysozyme by circular dichroism.

1. Human bronchial lysozyme was isolated from nonpurulent secretions and studied by circular dichroism (CD) spectroscopy for its conformational properties. 2. The two negative bands at 208 and 222 nm indicated that the peptide chain adopted an alpha-helical structure in physiological conditions. 3. The molecule was stable at pH 1.0 but not at pH 12.0. 4. Increasing ionic strength by adding NaCl up to 1 M did not change the CD spectra. 5. Complete unfolding of the molecule by guanidinium chloride was obtained only at the concentration of 6 M. 6. Bronchial lysozyme was also denatured by sodium dodecyl sulphate. 7. The molecule was stable when mild reduction was performed at 37 degrees C for 30 min but was completely unfolded after heating at 100 degrees C for 3 min.     

Secretion of lactoferrin and lysozyme by cultures of human airway epithelium

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.     

Toxic exoproducts of citrobacter freundii

A strain of Citrobacter freundii isolated from the feces of a patient with diarrhoea was examined for growth kinetics and toxic exoproduct formation using the complete (BHI) and synthetic culture media. It was found that the test organism in synthetic medium grew distinctly slower than in BHI. Fractionations on Sephadex G-100 column yielded 3 fractions from the complete medium culture filtrate and 2 fractions from the culture filtrate obtained from synthetic medium. The first culture filtrate fractions (F1) were represented by components of the molecular weight over 100,000, the respective second fractions (F2) from complete and synthetic medium were of the molecular weights of about 40,000 and 10,000. In the early skin test on rabbits the toxicity of culture filtrates and their fractions manifested itself by an increased permeability of blood vessels, in the late skin test by a hemorrhagic reaction associated with dilatation of blood vessels and induration of the skin tissue. In a test on mouse foot pad all separated filtrate fractions gave a positive edematous reaction. In cultured Vero cells samples of synthetic medium fractions gave a distinct cytotoxic reaction. Immunochemically, the presence of LPS in culture filtrates as well as some variations in the antigenicity of components from the complete and synthetic medium fractions were found. Apart from LPS some additional high-molecular-weight components were also present in the toxic complex of both first filtrate fractions (F1). Much more attention should be given to analysis of these first fraction complexes as well as to toxinogenicity of second fractions (F2) using some additional tests.     

Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.     

Inhibitory activity of a low molecular weight substance extracted from porcine small follicular fluid on estradiol and progesterone secretions by rat granulosa cells in vitro and by rat ovaries in vivo

Follicular fluid from small porcine follicles was filtrated through an Amicon XM-50 membrane to obtain a filtrate less than 50,000 MW. The filtrate was eluted through a Sephadex G-25 column (1.5 X 70 cm) using 0.01 N CH3COOH, pH 4.0, as elution buffer, and divided to five fractions. To test the inhibitory activity of these fractions on the in vitro estradiol and progesterone secretion, each fraction was added into a rat granulosa cell culture with FSH and testosterone in the medium. Two of five fractions exerted a significant inhibitory activity on the estradiol and progesterone secretions by the granulosa cells. They were in a range of low molecular weight fractions (MW 1,000-3,000) on the elution profile. Whether the in vitro active fractions are capable of inhibiting the in vivo estradiol and progesterone secretions by the ovary was assessed using the hypophysectomized DES-treated immature rat with hMG stimulation and the testosterone-treated immature rat with PMSG stimulation. The administration of the fractions to the former animal significantly suppressed the increases due to gonadotropin in the ovarian and serum estradiol concentrations. The administration of the fractions to the latter animal significantly suppressed the increases due to gonadotropin in the estradiol and progesterone concentrations of the ovary and serum. These results suggest that a low molecular weight substance from porcine small follicular fluid is capable of inhibiting the estradiol and progesterone biosyntheses in the follicle of the rat ovary.     

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.     

Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures.

Human skin procollagenase has been isolated, in pure form, from the medium of fibroblasts cultured in the presence or absence of added serum. Purification was achieved using a combination of cation-exchange (phosphocellulose or carboxymethylcellulose) and gel-filtration chromatography. Two forms (60 000 and 55 000 daltons) of the procollagenase were detected by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels and could be separated by chromatography on Ultrogel AcA-44. Each form was converted to active enzyme by trypsin, producing species of 50 000 and 45 000 daltons, respectively. An autoactivation process also occurred, which yielded active enzyme without a detectable change in molecular weight. Procollagenase also was found in organ cultures of human skin but only when serum was added to the medium. This suggests that a serum-inhibitable proteolytic system is present in these cultures which, like trypsin, converts procollagenase to the active enzyme forms that can be isolated from serum-free organ culture medium. The collagenase species obtained from either fibroblast or organ culture medium were chromatographically and electrophoretically identical.     

Characterization of lysozyme synthesized by differentiated mouse myeloid leukemia cells.

Lysozyme was induced by dexamethasone during normal differentiation of cultured mouse myeloid leukemia cells (M1) to macrophages and granulocytes. A large amount of lysozyme was produced by macrophage-like line cells (Mm-1), established from spontaneously differentiated macrophage-like cells from a clonal line of M1 cells. Lysozyme purified from the culture medium of these Mm-1 cells (Mm-1 lysozyme) had a molecular weight of 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and showed maximal activity at pH 6.6 with an optimal NaCl concentration of 0.04 M. Its mobility on polyacrylamide gel electrophoresis at pH 4.5 was distinctly lower than those of lysozymes from hen egg white and human urine. Rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-induced differentiated M1 cells, but not those of preparations from hen egg white and human urine. Lysozyme was also purified from normal mouse lung, which is rich in alveolar macrophages and was found to be similar to lysozyme purified from the culture medium of Mm-1 cells in size and electrophoretic mobility and in its pH optimum, trypsin peptide map, and antigenicity. Thus the molecular structure of the lysozyme induced in differentiated mouse myeloid leukemia cells is similar to that of lysozyme produced by normal cells.     

Preparation of lysozyme immobilized on textile carriers and its use in treating suppurative wounds

Immobilized forms of lysozyme were prepared by its covalent binding on dialdehyde cellulose and polycaproamide fibres as woven and knitted fabrics respectively. The preparations were estimated by the content of protein and bacteriolytic activity. The lysozyme activity per 1 g of the carrier and the protein content on dialdehyde cellulose were several times higher than those on polycaproamide while the specific activity of lysozyme on the polycaproamide carrier was somewhat higher than that on dialdehyde cellulose. The effect of the immobilized lysozyme in treatment of purulent wounds was studied on albino rats. It was shown that the periods of the wound healing with the use of the immobilized lysozyme were shorter than those with the use of native lysozyme. Cytological and morphological investigation of the wound wall confirmed the higher efficacy of the lysozyme immobilized forms in treatment of purulent wounds as compared to the use of the native enzyme.     

Calf rennet lysozyme.

A glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) was isolated from calf rennet. This lysozyme was also present in abomasal secretions from calf and adult cattle. Multiple molecular forms revealed by electrofocusing might be artefacts. The main enzyme form had Mr approx. 15 000, pH optimum 5.0, pI7.5, and a remarkable conformation stability. Competitive inhibition was observed with both N-acetylglucosamine and N-acetylmuramic acid, with apparent Ki values of 29 mM and 2.4 mM respectively. The isolated enzyme also displayed significant chitinase activity.     

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.     

Purification and characteriazation of collagenase from guinea pig skin.

Guinea pig skin col-agenase, isolated from culture medium of whole skin, was separated into two enzymatically active fractions. These two fractions have been purified extensively. Peak II fraction has been purified to homogeneity as examined by polyacrylamide gel electrophoresis. Their molecular weights are approximately 130 000 (peak I) and 40 000 (peak II). Both guinea pig skin collagenase fractions are capable of degrading the native collagen fibrils and are inhibited by serum, cysteine and EDTA. They appear to be glycoproteins. Guinea pig skin (peak II) and human skin collagenase were compared. They are both glycoproteins and have similar molecular size (Mr = 40 000). Immunodiffusion assay showed that no cross-reactivity was seen between the enzymes, indicating species specificity among collagenases.     

Purification of the basic protein avidin using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, from chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60-65% from one egg white with minimal lysozyme activity detected.     

Protein composition of human submandibular secretions

The protein composition of human submandibular saliva obtained from a single donor has been investigated, and 21 proteins have been resolved. On Sephadex G-100, submandibular secretions (unstimulated) separated into four fractions, I, II, III, and IV. Each fraction was analyzed further by isoelectric focusing and disc gel electrophoresis. The major components detected in each fraction along with their isoelectric point (pI) are as follows: I, blood group specific substance (2.3), immunoglobulin A (5.0–6.0), and immunoglobulin G (4.5–6.5); II, albumin (4.9), two glycoproteins (5.0), and acid phosphatase (5.2); III, three phosphoproteins (4.3–4.4), isoamylase 1A (5.9), isoamylase 1B (6.4), unidentified protein (7.1), lysozyme (>10), and a basic protein (>10); and IV, isoamylase 2A (5.9) and isoamylase 2B (6.4). Isoamylase 1A and IB are glycoproteins. Stimulated submandibular secretions were also resolved into four protein fractions by gel filtration. Fraction III, compared with unstimulated secretions, showed the greatest percent increase in protein. Analysis of this fraction by disc gel electrophoresis demonstrated the presence of four protein bands which were not detected in the unstimulated secretion. One of these proteins is tentatively identified as a phosphoprotein and two as basic proteins (pI > 10). The protein composition of submandibular, parotid, and sublingual secretions is compared.     

Isolation, purification, and partial chemical characterization of chromium(III) fractions existing in brewer's yeast and Sabouraud's liquid medium.

An ethanol extract of brewer's yeast which had been cultivated in a medium containing trivalent 51Cr was analyzed for 51Cr compounds by using petroleum ether extraction, gel filtration, cation and anion exchange chromatography and thin layer chromatography. Similar analytical procedures as for the above analysis were used for studying 51Cr compounds formed in the spent culture medium and in a sterile medium. Several 51Cr fractions were isolated from the three chromium sources, but one anionic 51Cr fraction present in the yeast and in the spent culture medium was not found in the sterile medium. Molecular weight estimations of the 51Cr fractions by gel filtration chromatography showed that the 51Cr ion exchange fractions contained several 51Cr compounds. The molecular weights of these compounds ranged from 150 to 1000 daltons and the molecular weights of 51Cr compounds separated from the yeast were markedly lower than those of the corresponding ion exchange fractions isolated from the culture medium. By using thin layer chromatography it was possible to isolate 51Cr compounds from the main bulk of ninhydrin active impurities. The polarity of all 51Cr compounds was found to be greater than that of most amino acids. The 51Cr compounds isolated from the yeast were mixed with 125I-insulin and incubated, after which the solution was eluted through Sephadex G-50 gel to test if binding had occurred. Elution peaks of 51Cr and 125I-insulin showed that 51Cr compounds were not bound to the insulin.     

Protein synthesis and degradation in a leucine auxotroph of Escherichia coli.

The synthesis and degradation of the soluble and sodium dodecyl sulfate-(SDS)-solubilized protein fractions of Escherichia coli were studied in both growing and nongrowing cultures. When separated according to molecular weight on SDS-polyacrylamide gels, the proteins of both fractions of growing cells undergo no measureable differential synthesis or degradation during logarithmic growth. However, when a leucine auxotroph is suspended in medium containing 5.3 muM leucine (a level that will not sustain growth), the SDS-solubilized protein of such a nongrowing culture shows a rapid synthesis of two protein components (32,000 and 12,000 daltons) found only in the out membrane.