Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution.
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 60kDa. 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.
Jayakumar
Arumugam
A
Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4009, USA.
Kang
Ya'an
Y
Henderson
Ying
Y
Mitsudo
Kenji
K
Liu
Xiaoling
X
Briggs
Katrina
K
Wang
Mary
M
Frederick
Mitchell J
MJ
El-Naggar
Adel K
AK
Bebök
Zsuzsa
Z
Clayman
Gary L
GL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017353:Gene Deletion; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000502:physiology*; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins / Q000378:metabolism; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism*
D002352:Carrier Proteins; D021382:Protein Sorting Signals; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5