Proteolytic processing of human growth hormone by multiple tissue kallikreins and regulation by the serine protease inhibitor Kazal-Type5 (SPINK5) protein.
BACKGROUND
Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro.


METHODS
Expression of 12 of the KLKs (KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities.


RESULTS
We here describe for the first time expression of numerous KLKs (KLKs 5-8, 10-14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4-6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro. Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro. Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation.


CONCLUSIONS
We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.
Komatsu
Nahoko
N
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1L5.
Saijoh
Kiyofumi
K
Otsuki
Norio
N
Kishi
Tadaaki
T
Micheal
Iacovos P
IP
Obiezu
Christina V
CV
Borgono
Carla A
CA
Takehara
Kazuhiko
K
Jayakumar
Arumugam
A
Wu
Hua Kang
HK
Clayman
Gary L
GL
Diamandis
Eleftherios P
EP
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D010446:Peptide Fragments / Q000494:pharmacology; D053491:Proteinase Inhibitory Proteins, Secretory; D012333:RNA, Messenger / Q000235:genetics; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D020840:Tissue Kallikreins / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D010446:Peptide Fragments; D053491:Proteinase Inhibitory Proteins, Secretory; D012333:RNA, Messenger; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D019382:Human Growth Hormone; D020840:Tissue Kallikreins