Sikandar L. Katyal

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein.

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.

Clara cell 10 kDa protein (CC10): Comparison of structure and function to uterogloblin

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.

Clara cell proteins.

Abstract: Clara cells are nonciliated, nonmucous, secretory cells of the pulmonary airways. These cells are known to secrete a variety of proteins, including Clara cell 10‐kDa protein/uteroglobin. This protein consists of a homodimer of 70–77 amino acid polypeptides arranged in antiparallel fashion. In vitro testing suggests that the protein suppresses inflammation. The physiologic role of the protein remains to be determined.

An immunologic study of the secretory products of rat Clara cells

Lungs of adult rats were lavaged with normal saline containing 0.25 mM phenylmethylsulfonyl fluoride. The surfactant pellet was removed by centrifugation and serum proteins in the lavage were removed by affinity chromatography using rabbit anti-rat whole serum antiserum. The residual proteins, thought to represent products of secretory cells, were used as the immunogen to inject rabbits. The resulting antiserum was absorbed with affinity columns of rat serum and rat liver extract. The gamma globulin fraction of the unbound antiserum was found to react with two proteins in the lavage by immunodiffusion and crossed immunoelectrophoresis. The antiserum specifically stained, by the immunoperoxidase method, a subpopulation of cells consistent in morphology with Clara cells lining the bronchioles and bronchi. The antigens were detectable, by immunohistochemistry, in rat fetus at 19 days of gestation, a progressive increase in the antigen content was noted with increasing gestational age and an adult pattern was noted at 2 weeks of age. In adult animals the intracellular content of the antigens appears to be about twofold greater than their content in the lavage fluid.

Expression of HGF and cMet in the developing and adult brain

Hepatocyte growth factor (HGF) was recently recognized as a potential neurotrophic factor in the developing brain. We studied expression of HGF and its receptor using Northern blot analysis and in situ hybridization for mRNA and double immunofluorescent laser confocal microscopy. HGF and cMet messages were abundant in the hippocampus of both human and rat brains. In this region, both messages were localized in the neuronal layer. Segregation of HGF predominantly in the hippocampal CA3-4 and cMet in CA1 supports the hypothesis that HGF may mediate important neurotrophic functions in both developing and adult brains.

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.

Analysis of pulmonary surfactant apoproteins by electrophoresis

Surfactant apoproteins were prepared from detergent-solubilized rat surfactant by immunoaffinity chromatography. SDS-polyacrylamide gel electrophoresis of the apoproteins, without prior chemical reduction, revealed several bands of molecular weights 50 000-78 000, 140 000 and 160 000. Following treatment with dithiothreitol, the apoproteins were resolved into three bands of molecular weights 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 daltghts 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 daltghts 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 dalton protein appeared to be the richest in carbohydrates, followed by the 32 000 and 26 000 dalton proteins. Partial proteolysis of the 38 000 and 32 000 dalton proteins showed similarity in the sizes of peptides generated. Surfactant-associated proteins from human and monkey lungs were also analyzed by polyacrylamide gel electrophoresis. A non-serum glycoprotein of molecular weight 38 000 was found. In different systems of polyacrylamide gel electrophoresis, this protein showed an electrophoretic mobility similar to that of the 38 000 dalton protein of rat surfactant. However, it formed polymers of molecular weight higher than those of polymers found in rat surfactant.

Choline Deficiency and Chemical Carcinogenesis

We have reviewed the current status of our knowledge concerning the biologic effects of dietary choline (lipotrope) deficiency in modifying chemical carcinogenesis in experimental animals and discussed its possible mechanisms. Choline deficiency produces various pathologic lesions, involving virtually every organ of the body, as a result of a decrease in phospholipid and acetylcholine synthesis and in the supply of labile methyl groups. The liver is the only organ in which a relationship has been consistently demonstrated between choline deficiency and chemically induced tumors. The deficient diet enhances the initiating potency of several carcinogens and acts as a strong cocarcinogen. Diet also exerts a strong promoting effect, though the possibility that it is a complete carcinogen cannot be ruled out. Phase I enzymes of the carcinogen metabolizing system are uniformly depressed by choline deficiency, but very little information is available regarding the effects of diet on Phase II enzymes that detoxify carcinogen metabolites. Possible modifications of carcinogen-induced DNA damage and their repair processes have not been adequately scrutinized. Solid evidence suggests that feeding a choline-deficient diet leads to enhanced liver cell proliferation, an inadequate supply of methyl groups for transmethylation reactions, and membrane lipid peroxidation. Induced cell proliferation and hypomethylation of DNA may alter the state of gene expression, including that of specific cellular oncogenes. Lipid peroxidation may alter the structure and function of membrane receptors related to liver cell growth or may directly damage cellular DNA. Thus these alterations, individually or in combination, could play a critical role in the diet-induced modification of chemical carcinogenesis.

Papillary adenoma of type 2 pneumocytes

A case of papillary adenoma of type 2 pneumocytes is reported. A 57-year-old man had an unusual coin lesion in the periphery of the right lung without any symptoms. When detected in a mass survey examination, it was approximately 1.5 cm in diameter, well circumscribed, and located in S4, involving the sixth-order bronchus of B4. Light-microscopic examination revealed cuboidal tumor cells arranged in a papillary pattern. Ultrastructurally, the cells had characteristic osmiophilic lamellar bodies. By immunoperoxidase staining, the tumor cells were shown to have intracytoplasmic surfactant apoproteins. The postoperative course was uneventful, and there is no evidence of disease 8 years later.

Analysis of pulmonary surfactant apoproteins by isoelectric focusing

Apoproteins of Mr 38 000, 32 000 and 26 000 are found in surfactant isolated from rat lungs. The surfactant isolated from monkey lungs, on the other hand, contains the 38 kDa apoprotein and not the 32 and 26 kDa apoproteins. These preparations of pulmonary surfactant contain, in addition, several serum proteins. We have used a combination of salt- and sucrose-density gradient centrifugations to isolate and further purify surfactant from the washings of rat lungs. Thus, a preparation of pulmonary surfactant was obtained which contained exclusively the 38, 32, 26 and 10-12 kDa apoproteins, and which was rich in phosphatidylcholine and phosphatidylglycerol. Using an immunoassay and an immunoblotting technique, it was established that the 38, 32 and 26 kDa apoproteins are not serum proteins. The surfactant apoproteins of rat and monkey were further subjected to the high-resolution of isoelectric focusing. Thus, rat surfactant apoproteins resolved into 11 bands in the pH range 4.64-5.53. A second-dimensional electrophoresis in a sodium dodecyl sulfate system led to the migration of the 11 bands, separated by first-dimensional isoelectric focusing, into three distinct groups with apparent molecular weights of 38 000, 32 000 and 26 000, respectively. Upon isoelectric focusing, the apoproteins from monkey lung surfactant also separated into several bands in the pH range 5.18-5.82. After electrophoresis in the second dimension as above, these bands migrated as a single group with an apparent molecular weight of 38 000. Neuraminidase treatment of rat surfactant apoproteins, and subsequent IEF, led to the disappearance of several low-pI variants with a concomitant increase in the amounts of higher-pI variants. Thus, the sialic acid content of surfactant apoproteins accounts for, in large part, the observed charge heterogeneity.