TY - JOUR
T1 - Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution
AU - Jayakumar, Arumugam
AU - Kang, Ya'an
AU - Henderson, Ying
AU - Mitsudo, Kenji
AU - Liu, Xiaoling
AU - Briggs, Katrina
AU - Wang, Mary
AU - Frederick, Mitchell J.
AU - El-Naggar, Adel K.
AU - Bebök, Zsuzsa
AU - Clayman, Gary L.
N1 - Funding Information:
This work was supported in part by the University of Texas M.D. Anderson Cancer Center SPORE in Head and Neck Cancer NIH-NCI P50 CA097007 (G.L.C. and M.J.F.), NIH R01 DE013954 (G.L.C.), Cancer Center Support Grant NIH P30 CA016672 (G.L.C), Alando J. Ballantyne Distinguished Chair in Head and Neck Surgery award (G.L.C.), Michael A. O’Bannon Endowment for Cancer Research (G.L.C.), Betty Berry Cancer Research Fund (G.L.C.), and NIH INRS Award T32 CA060374 (G.L.C.), and 2004 AAO-HNSF Percy Memorial Grant (G.L.C.). The work of K.M. was partly supported by a Grant-in-aid for Scientific Research (C) (No. 16591991) from Ministry of Education, Culture, Sports, Science and Technology in Tokyo, Japan.
PY - 2005/3/1
Y1 - 2005/3/1
N2 - The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60 kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion.
AB - The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60 kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion.
KW - Endoplasmic reticulum
KW - Furin
KW - HNSCC
KW - LEKTI1-6
KW - Pro-LEKTI
KW - Signal peptide
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U2 - 10.1016/j.abb.2004.12.012
DO - 10.1016/j.abb.2004.12.012
M3 - Article
C2 - 15680911
AN - SCOPUS:19944432497
SN - 0003-9861
VL - 435
SP - 89
EP - 102
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -