program focuses on airway cell biology with a main emphasis on the function
and composition of secretions in normal and diseased airways. The research
area provides opportunities for graduate students and postdoctoral fellows
with diverse interests to develop their own research programs. A variety of techniques are available for use on projects
including recombinant DNA/molecular biology, immunochemistry, various types
of microscopy, cell culture and subcellular fractionation.
Conner, G. E., C. Wijkstrom-Frei, S. H. Randell, V. E. Fernandez and M. Salathe.
The lactoperoxidase system links anion transport to host defense in cystic
letters 581: 271-278 (2007)
Schmid, A., G. Bai, N.
Schmid, M. Zaccolo, L. E.
Ostrowski, G. E. Conner, N. Fregien
and M. Salathe. Real-time analysis of cAMP-mediated
regulation of ciliary motility in single primary
human airway epithelial cells. J
Cell Sci 119: 4176-4186 (2006)
Gerson, C., J. Sabater,
M. Scuri, A. Torbati, R.
Coffey, J.W. Abraham, I. Lauredo, R. Forteza, A. Wanner, M. Salathe, W. M. Abraham, and G. E. Conner. The
Lactoperoxidase System Functions in Bacterial Clearance of Airways. Am.
J. Resp. Cell Mol. Biol. 22: 665-671, 2000
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 serves 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
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 anpseudostratified 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 this movie.
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.
interface cultures of airway epithelial cells showing ciliated cells and
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
Thiocyanate and Cystic Fibrosis
Since lactoperoxidase requires an anion for its activity, thiocyanate transport defects might provide a link
between the defective anion channel (CFTR) in cystic fibrosis and chronic
airway infection that is characteristic of this disease. We demonstrated
defective thiocyanate transport in cystic
fibrosis airway epithelia (Am. J. Respir. Crit.
Care Med. 163: A490 (2001); J.
Physiol. 561: 183-194 (2004);
FEBS Letters 581: 271-278 (2007)) supporting the idea that a loss of thiocyanate may contribute to increased infection
through lack of lactoperoxidase activity. Current studies are examining the
actual levels of thiocyanate in normal and CF
It is interesting to note that compuational
modeling of airway surface liquid (FEBS
Letters 581: 271-278 (2007)) suggests that CF airways might have
increased hydrogen peroxide due to a loss of lactoperoxidase activity as
lactoperoxidase is normally the major consumer of hydrogen peroxide in
airway secretions (Am
J RespirCrit Care Med
167: 425-430 (2003)). Elevated hdyrogen
peroxide may contribute to the chronic inflammation seen in CF.
Human fibroblast cathepsin D cDNAs pCPSD1 -full
length preprocathepsin D with 80 nt 5' UTR, expressibleinmamalian cells pTCPSD - procathepsin D
without the signal peptide in a pET vector for
pVL1392CD- preprocathepsin D for homologous
recombination using bacculovirus
Human fibroblast procathepsin D antiserum and recombinat human procathepsin
Here are the most frequently requested cathepsin
Scarborough, P.E., K. Guruprasad, C. Topham, G. icho, 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