AIRWAY CELL BIOLOGY

Protection of the Respiratory System
Secreted Enzymes of the Airway Mucosa

        The airway mucosa represents one of the most important interfaces between an animal and its environment. The mucosa must provide a sophisticated defense against airborne material of a variety of sizes and composition. Unsuccessful or inappropriate response of the airway mucosa is detrimental to the airway and underlying tissues.  Our program in pulmonary cell biology studies components of airway secretions that may play a role in respiratory diseases.

        Hydrogen peroxide has been shown by others to be elevated during airway inflammatory diseases such as asthma and is a major contributor to the inflammatory reactions associated with a variety of airway diseases.  Our studies have identified the major hydrogen peroxide scavenging activity in airway secretions. We have now purified and characterized the activity and shown it to be a single protein that comprises 1% of the soluble secreted protein in sheep airways. Sequence analysis of the purified peroxidase together with cDNA cloning and enzymatic and spectral analysis show that the peroxidase is identical to lactoperoxidase (LPO) expressed in mammary gland. Airway LPO is secreted by goblet cells and functions both in a biocidal capacity and in controlling the reactive oxygen species in the airway.  Other biochemically similar peroxidases produce biocidal compounds to protect against infection. We have shown that airway peroxidase has a similar function in the sheep and human respiratory tract. 

        Human airway secretions also contain peroxidase and we are studying levels of  airway peroxidase and substrates in secretions of cystic fibrosis patients who have frequent bacterial infections and who have defects in ion transport that may also alter peroxidase substrate concentrations. We are also studying peroxidase in asthmatics who typically have elevated levels of hydrogen peroxide during periods of heightened inflammation.  In addition,  we are examining the regulation of the peroxidase system in cultures of differentiated airway epithelial cells. Finally we are working on the molecular sources of hydrogen peroxide in airway epithelia.

    Tissue kallikrein is also secreted into the airway primarily by serous cells of the submucosal glands. This enzyme cleaves polypeptide hormones and precursors into active forms. Bronchial tissue kallikrein mediates bronchoconstriction in response to a variety of stimuli by cleaving high molecular weight kininogen to form lysyl-bradykinin that in tern causes bronchial smooth muscle contraction. Bronchial kallikrein is believed to mediate aspects of airway hyperresponsiveness and airway inflammation and thus is thought to play a role in asthma. This enzyme is currently being purified and characterized in order to better understand how it is regulated in normal and inflamed airways.

      Hyaluronan is also secreted into the airway lumen and server mutiple functions. Besides contributing to the viscoelastic properties of mucus, it also regulates enzymatic activity of tissue kallikrein and serves to immobilize kallikrein and airway LPO on the surface epithelial cells. This immobilization is mediated by hyaluronan binding to cell surface RHAMM and prevents the removal of the enzymes by the constant ciliary movement that clears the airways.

        We use human airway epithelial cells growing in culture at an air-liquid interface as a model system. Cells are harvested from human lungs obtained from the Organ Procurement Organization through local IRB approved protocols. After dissection, airway mucosa is digested with proteases and cells are plated in normal submerged culture for expansion. For experiments, cells are plated on transwells consisting of a filter support that allows manipulation of separate apical and basolateral compartments. After growth to confluency, media is removed from the apical surface and the culture differentiates into an pseudostratified columnar epithelial layer with ciliated cells and goblet cells readily apparent. These cultures are then used to study synthesis and secretion of mucus components, transepithelial transport, ciliary beat frequency and other features specific to airway epithelia.

The beating of cilia on the surface of the cultures moves secreted mucus and trapped debris in a circular pattern reflecting the geometry of the culture system. These circular movements resemble "hurricanes" and can be seen in the quicktime movie to the right.

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Cytochemical detection of airway peroxidase. Fixed sheep trachea were incubated in diaminobenzidine and hydrogen peroxide (panel a) or diaminobenzidine alone (panel c). The dark precipitated product of the airway peroxidase is visible in (arrow inpanel a). The same section in panel a was stained to show goblet cells in panelb.

Air-liquid interface cultures of airway epithelial cells showing ciliated cells and goblet cells.

Reactive Oxygen Species in the Airway

The Dual Oxidases (Duox 1 and Duox 2) have been identified as a major source of reactive oxygen species (ROS) including hydrogen peroxide in the airway. Lactoperoxidase has been identified as the major scavenger of hydrogen peroxide in airway secretions. Since lactoperoxidase requires hydrogen peroxide for its antibacterial activity, we are exploring the regualtion of  Duox activity in the airway. We have shown that intracellular calcium concentrations regulate the activity of Duox in airway epithelia using a real-time fluorescence measurement of hydrogen peroxide production in response to stimuli that increase intracellular calcium, e.g. purinergic receptor stimulation. We are also studying the intracellular location of the Duox forms and have found that only a small portion of Duox is exposed at the cell surface

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Structure and Function of Cathepsin D
       
Look here for  structural, enzymatic, or genetic  info on Cathepsin D.

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Cathepsin D Reagents and Protocols
Available Reagents (Contact info):

Human fibroblast cathepsin D cDNAs
    pCPSD1 -full length preprocathepsin D with 80 nt 5' UTR, expressiblein mamalian cells
    pTCPSD - procathepsin D without the signal peptide in a pET vector for bacterial expression
    pVL1392CD- preprocathepsin D for homologous recombination using bacculovirus

Human fibroblast procathepsin D antiserum and recombinat human procathepsin inclusion bodies.

Here are the most frequently requested cathepsin D protocols:

    Immunoprecipitation using our polyvalent antibodies against human fibroblast procathepsin D: -an example of immunoprecipitates from human endothelial cells .

    Pepstatin Agarose Purification of  Cathepsin D and Procathepsin D including tissues, cultured cells and media
            Assay and Activation
           Pepstatin Agarose Preparation

 SOME CATHEPSIN D PUBLICATIONS:

Conner, G.E., J.A. Udey, C. Pinto, and J. Sola. Nonhuman Cells Correctly Sort and Process the Human Lysosomal Enzyme Cathepsin D. Biochemistry 28: 3530-3533, 1989. 

Conner, G.E. Isolation of Procathepsin D from Cathepsin D by Pepstatin Affinity Chromatography:Autocatalytic Conversion of an inactive to active form of the enzyme. Biochem. J. 263: 601-604, 1989.

Conner GE, Udey JA. Expression and refolding of recombinant human fibroblast procathepsin D. DNA Cell Biol 9:1-9, 1990

Conner, G.E. The Role of the Procathepsin D Propeptide in Sorting to the Lysosome. J. Biol. Chem. 267: 21738-21745, 1992.

Scarborough, P.E., K. Guruprasad, C. Topham, G. Richo, G.E. Conner, T.L. Blundell and B.M. Dunn. Exploration of Subsite Binding Specificity of Human Cathepsin D through Kinetics and Rule-based Molecular Modelling. Protein Science 2: 264-276, 1993

Zhu, Y. and G.E. Conner. Intermolecular Association of Lysosomal Protein Precursors During Biosynthesis. J. Biol. Chem. 269: 3846-3851, 1994

Richo, G. and G.E. Conner. Structural Requirements of Procathepsin D Activation and Maturation. J. Biol. Chem. 269: 14806-14812, 1994

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