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Human Molecular Genetics, 2002, Vol. 11, No. 3 301-315
© 2002 Oxford University Press

Synthesis, purification and structural and functional characterization of recombinant form of a common genetic variant of human luteinizing hormone

Pulak R. Manna, Lata Joshi, Vernon N. Reinhold2, Michel L. Aubert3, Nobuhiko Suganuma4, Kim Pettersson1 and Ilpo T. Huhtaniemi+

Department of Physiology and 1Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland, 2Department of Chemistry, University of New Hampshire, Durham, NH 03824-3598, USA, 3Department of Pediatrics, University of Geneva, 1211 Geneva 14, Switzerland and 4Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Handa 475, Japan

A common genetic variant (V) of luteinizing hormone (LH), with two mutations (Trp8Arg and Ile15Thr) and an extra glycosylation consensus site (Asn13-Ala-Thr), is associated with abnormalities of reproductive function. To address the molecular basis of the functional differences between V- and wild-type (WT)-LH, recombinant (rec) forms of WT- and V-LH were synthesized in human embryonic kidney (HEK 293) cells. The rec hormones synthesized were rigorously purified employing affinity, immunoaffinity and ion exchange chromatographies (final purity ~12 000 IU/mg, 180-fold purification, 28% recovery). Functional properties of the hormone preparations were compared in vitro and in vivo. The molecular size of both rec LHs was 31 kDa, as determined by SDS–PAGE. Although the mutations in V-LHß did not significantly affect the affinity of LH receptor (LHR) binding (Kd ~0.4 nmol/L), V-LH had higher in vitro biopotency than WT-LH, in terms of mLTC-1 mouse Leydig tumor cell cAMP and progesterone (P) production, and steroidogenic acute regulatory protein (StAR) expression. In addition, in HEK 293 cells expressing the human LHR, V-LH demonstrated 1.8-fold higher response of inositol trisphosphate (IP3) production than WT-LH. Furthermore, HEK 293 cells expressing the ElK1 trans-reporting plasmids displayed 2.7-fold greater luciferase response to V-LH than WT-LH, documenting stimulation of the mitogen-activated protein kinase (MAPK) pathway. The in vivo half-life of V-LH was clearly faster (5–9 min) than that of WT-LH (12–22 min) and human chorionic gonadotropin (hCG; 50–70 min), when injected into rat circulation. It is worth noting that analysis by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS) demonstrated clear differences in structures of carbohydrate side chains attached to the two forms of rec LHs, including incomplete processing of high mannose glycans (Man5,8,9) in V-LH, suggesting different pathways in its intracellular trafficking. Collectively, the present findings provide the molecular basis for the qualitative and quantitative differences in LH action that are observed in carriers of the V-LHß allele.

+ To whom correspondence should be addressed at: Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel: +358 2 3337579; Fax: +358 2 2502610; Email: ilpo.huhtaniemi@utu.fi The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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