Jane D. Funkhouser

Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family

BackgroundChitinases (EC.3.2.1.14) hydrolyze the β-1,4-linkages in chitin, an abundant N-acetyl-β-D-glucosamine polysaccharide that is a structural component of protective biological matrices such as insect exoskeletons and fungal cell walls. The glycoside hydrolase 18 (GH18) family of chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Mammals are not known to synthesize chitin or metabolize it as a nutrient, yet the human genome encodes eight GH18 family members. Some GH18 proteins lack an essential catalytic glutamic acid and are likely to act as lectins rather than as enzymes. This study used comparative genomic analysis to address the evolutionary history of the GH18 multiprotein family, from early eukaryotes to mammals, in an effort to understand the forces that shaped the human genome content of chitinase related proteins.ResultsGene duplication and loss according to a birth-and-death model of evolution is a feature of the evolutionary history of the GH18 family. The current human family likely originated from ancient genes present at the time of the bilaterian expansion (approx. 550 mya). The family expanded in the chitinous protostomes C. elegans and D. melanogaster, declined in early deuterostomes as chitin synthesis disappeared, and expanded again in late deuterostomes with a significant increase in gene number after the avian/mammalian split.ConclusionThis comprehensive genomic study of animal GH18 proteins reveals three major phylogenetic groups in the family: chitobiases, chitinases/chitolectins, and stabilin-1 interacting chitolectins. Only the chitinase/chitolectin group is associated with expansion in late deuterostomes. Finding that the human GH18 gene family is closely linked to the human major histocompatibility complex paralogon on chromosome 1, together with the recent association of GH18 chitinase activity with Th2 cell inflammation, suggests that its late expansion could be related to an emerging interface of innate and adaptive immunity during early vertebrate history.

Monoclonal antibody identification of a type II alveolar epithelial cell antigen and expression of the antigen during lung development

A monoclonal antibody identifying an antigen expressed by rat type II alveolar epithelial cells, but not by type I epithelial cells or other mature lung cells, was produced by immunization of mice with cells of the rat L2 cell line. The antigen recognized by the antibody was present on the microvillous luminal surface of type II epithelial cells. In adult rat lung, only type II epithelial cells bound the antibody. During fetal development the antigen was expressed by cuboidal epithelial cells lining the respiratory ducts of the first divisions of the tracheal bud, but not by epithelial cells lining the esophagus or trachea. The antigen continued to be expressed by cuboidal epithelial cells lining the larger respiratory ducts until approximately 19 days gestational age. Thereafter, expression was increasingly limited to selected single cells or clusters of two to four cuboidal cells in the smallest ducts. By the 21st postnatal day, the antigen was expressed only by type II alveolar epithelial cells. Type II alveolar epithelial cells isolated from adult lung and the L2 cell line in culture expressed the antigen on the cell surface. A protein of approximately 146,000 Mr was isolated by immunoadsorption of the antigen from non-ionic detergent extracts of type II cells and L2 cells. Preliminary studies of the binding of the antibody to other rat tissues indicate that the antibody binds to renal proximal tubular epithelial cells of the kidney and the luminal surface of the small bowel epithelial cells.

An organ culture system for study of fetal lung development.

Abstract Fetal rat lungs placed in in vitro organ culture at 15.5 days gestation grow significantly based on accumulation of DNA and protein. In the experimental system described, DNA accumulated rapidly during the first three days in culture and increased from 4.8 to 15.6 micrograms per lung culture. Protein content increased more slowly and reached a value more than double the initial value after six days in the culture system. Glycogen accumulated in the tissue during the first six days in culture and was depleted during the subsequent culture period, a pattern strikingly similar to that observed during lung development in vivo . Phospholipid accumulation was biphasic with respect to time with an inflection point at about the sixth day of culture. The phosphatidylcholine species synthesized in the culture system in vitro were similar to those produced in vivo in fetal lung at 21 days gestation.

Properties of a non-specific phospholipid-transfer protein purified from rat lung.

The present report describes the purification and characterization of a non-specific phospholipid-transfer protein from rat lung. The protein is the major phospholipid-transfer protein in lung which transfers phosphatidylcholine. The transfer protein was purified 1200-fold, with a final yield of 3%. The activity of the protein was monitored by measuring the transfer of [14C]phosphatidylcholine from radioactively labeled liposomes to mitochondria. The purified proteins transfers phosphatidylcholine, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine from radioactively labeled microsomes to either mitochondria or liposomes. The transfer of each phospholipid is proportional to its content in the donor membrane. The protein was purified from a pH 5.1 supernatant preparation by fractionation on DEAE-cellulose, Sephadex G-75 and hydroxyapatite. The molecular weight of the purified protein was estimated as 35 000 by SDS-polyacrylamide gel electrophoresis. The amino acid analysis revealed a high content of glutamic acid (including glutamine) and glycine. The specificity of the purified protein for transfer of phospholipids suggests that it may be the phospholipid-transfer activity which is highly enriched in isolated type II alveolar cells of rat lung.

Speculations on ataxia-telangiectasia: defective regulation of the immunoglobulin gene superfamily

In this short article, Raymond Peterson and Jane Funkhouser develop the argument that the common molecular mechanism linking the various clinical manifestations of ataxia-telangiectasia (AT) is a defect in the regulation of the immunoglobulin (Ig) gene superfamily. They propose that the AT gene codes for a protein essential for the orderly expression of this gene family, perhaps regulating the gene rearrangement process that appears to be a unique characteristic of this system. Members of the Ig gene superfamily play a major role in the development and operation of the immune and nervous systems, and any perturbation of their expression would be anticipated to produce a panoply of signs and symptoms, such as those characterizing the AT phenotype.

Glucocorticoids and fetal lung development

Abstract Acceleration of fetal lung development by administration of glucocorticoid hormones has been demonstrated in a number of mammalian species. Dexamethasone 10 −5 M significantly depressed protein content per explant in the presence and absence of insulin. Insulin did not alter the protein content but significantly increased radioactive choline incorporation. However, dexamethasone provided no added stimulation above that observed with insulin alone. The incorporation of radioactive choline and specific activities in isolated phosphatidylcholine were almost identical in control and dexamethasone (10 −7 M) treated explants. The data presented demonstrate a modest but significant increase in choline incorporation using a concentration of dexamethasone of 10 −9 M. At higher concentrations of dexamethasone, the incorporation was significantly depressed using an 8 day exposure time. Although choline incorporation increases as a function of gestational age with a burst in rate on the last day of fetal life, dexamethasone suppresses this activity at all developmental ages past 19.5 days. The only conclusion that appears valid at this time is the rat fetal lung system reported here differs in an unknown way when compared to the rabbit monolayer system, the human organ culture, and the in vivo situation.

Acyl-chain specificity and membrane fluidity: Factors which influence the activity of a purified phospholipid-transfer protein from lung

A purified phospholipid-transfer protein from rat lung has been characterized in terms of the specificity of the protein for phosphatidylcholine molecules with different apolar moieties. The study demonstrated that the lung-phospholipid-transfer protein discriminates between dipalmitoylphosphatidylcholine and molecular species of phosphatidylcholine with unsaturated acyl chains. The initial rate of transfer of dipalmitoylphosphatidylcholine is 1.5-fold greater than the rate of transfer of dioleoylphosphatidylcholine, 1-palmitoyl-2- arachidonylphosphatidylcholine , or egg phosphatidylcholine under most assay conditions. Although the protein preferentially transfers dipalmitoylphosphatidylcholine, the incorporation of increasing mole percentages of dipalmitoylphosphatidylcholine into unilamellar phosphatidylcholine vesicles profoundly affects their effectiveness as donors for phosphatidylcholine transfer by the transfer protein. At 60 mol% dipalmitoylphosphatidylcholine, the rate of transfer is one-third that observed when vesicles are composed of 100% egg phosphatidylcholine. Decreases in membrane fluidity as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene correlate with decreases in the effectiveness of the vesicles as donors in the phospholipid-transfer reaction. The conclusion from these studies is that the rate of transfer of phosphatidylcholine by the purified phospholipid-transfer protein from lung is determined by physical properties of membrane interfaces with which the protein interacts, as well as by the specificity of the phospholipid-transfer protein for different molecular species of phosphatidylcholine.

Phospholipid transfer proteins from lung, properties and possible physiological functions

Phospholipid transfer proteins have been found in lung just as they have in tissues throughout the body. There is speculation that the proteins are involved in membrane biogenesis and in determining the phospholipid composition of membranes. For this reason the lung, which contains subcellular organelles of distinct phospholipid composition, is of interest in terms of its complement of phospholipid transfer proteins. The lamellar bodies of pulmonary type II alveolar cells have a phospholipid composition unique in terms of the proportions of dipalmitoyl phosphatidylcholine and phosphatidylglycerol. Studies of the phospholipid transfer proteins in lung have demonstrated two molecular species of the transfer proteins that differ significantly from those found in liver and other tissues. These proteins show specificity for the transfer of dipalmitoyl phosphatidylcholine and phosphatidylglycerol.

Fetal lung disaturated phosphatidylcholine: Ostensible increase following exposure to dexamethasone

Fetal rat lung removed at 15 days gestation and placed in organ culture incorporates choline into phosphatidylcholine. Addition of 10(-9) M dexamethasone resulted in increased rates of choline incorporation per micrograms protein after both 6 and 12 days culture. This concentration of dexamethasone did not increase tissue phosphatidylcholine or disaturated phosphatidylcholine. Thus, at a culture time when dexamethasone had a significant effect on choline incorporation, there was no change in either the total phospholipid or disaturated phosphatidylcholine content of the lung tissue. The transplacental administration of dexamethasone decreased fetal lung DNA and phospholipid content. At the mid-range dosage tested (400 micrograms), dexamethasone depressed DNA (51%) appreciably more than total phosphatidylcholine (28%) and disaturated phosphatidylcholine (33%). These results show that the hormone does not increase the total amount of surfactant per lung. The increased disaturated phosphatidylcholine per mg DNA results in an ostensible beneficial effect of dexamethasone on surfactant and may reflect an increased proportion of Type II cells in fetal lung both in vitro and in vivo following hormone exposure. Disaturated phosphatidylcholine per Type II alveolar cell is no doubt increased but the trade-off is fewer total cells in the lung.

The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer.

Lung lamellar bodies and liver mitochondria were used to demonstrate that soluble phospholipid transfer proteins from lung transfer phosphatidylcholine to both of these acceptors. The initial rate of transfer to lung lamellar bodies is about half that of the rate of transfer to the liver mitochondria when both acceptor membranes are present at saturating concentrations. Phosphatidylcholine unilamellar vesicles were used to demonstrate that the fatty acyl composition of the membrane phosphatidylcholine is a significant determinant of the rate of phosphatidylcholine transfer catalyzed by these proteins. The lamellar bodies have a unique phosphatidylcholine composition, and these studies suggest that this is an important factor in determining the lower initial rate of transfer to lamellar bodies. The studies have also characterized two phospholipid transfer proteins in rat lung in terms of isoelectric point. Isoelectric points for the two proteins which transfer phosphatidylcholine were found to be 5.6 +/- 0.08 and 6.2 +/- 0.03.