Description and Characterisation of a Splicing-Variant of Insulin-Like Growth Factor-Binding Protein-7 (IGFBP-7) Expressed in Human Granulosa Cells

Zusammenfassung

Fragestellung und Methodik

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPS) sind kürzlich beschriebene zysteinreiche Proteine, die am aminoterminalen Ende ausgeprägte Strukturhomologien zu den bisher beschriebenen IGFBPs haben. IGFBPs sind in einer Vielzahl biologischer Funktionen involviert, einschließlich der Wachstumsregulierung und der Anheftung endothelialer Zellen. Ihre Wirkung können sie sowohl in Abhängigkeit von IGF als auch unabhängig von IGF entfalten. Im Verlauf eines Projektes zur Identifizierung und Charakterisierung von angiogenetisch wirksamen Faktoren haben wir eine Spleißvariante von IGFBP-rP-1/IGFBP-7 identifiziert (IGFBP-7 v) und charakterisiert, die in humanen Primärkulturen von Granulosazellen exprimiert wird, aber nicht in Tumorzelllinien, die von Ovarial-, Brust- und Zervixkarzinomen abgeleitet sind (HeLa, SW756; Caski; Ovcar-3, MDA-MP453).

Ergebnisse

IGFBP-7 v ist um 165 Nukleotide im ersten Exon verkürzt, was einem um 55 Aminosäuren verkürztem Protein entspricht. Diese Deletion umschließt das IGFBP-typische Aminosäure-Motiv, welches für die Bindung von IGF verantwortlich gemacht wird.

Schlussfolgerung

Wir vermuten daher, dass diese natürlich vorkommende Spleißvariante von IGFBP-7 unabhängig von der Bindung von IGF wirken kann.

Abstract

Objective

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPs) are recently described cysteine-rich proteins that share significant amino terminal structural similarity with the conventional IGFBPs and are involved in a variety of biological functions, including growth regulation and endothelial cell attachment and spreading. In an effort to screen for angiogenic factors in human granulosa cells (GC), we have identified and characterised a splice variant of IGFBP-7/IGFBP-rP-1 (IGFBP-7 v).

Methods

The presence of IGFBP-7 and IGFBP-7 v variant was detected by RT-PCR in human granulosa cells derived from women undergoing in vitro fertilisation therapy. The nucleotide sequence was determined after subcloning and sequencing according to standard techniques. Computer analyses were performed at the ExPASy Molecular Biology server using the ClustalW alignment and protein analysis programms (http://www.expasy.ch).

Results and Conclusions

The splice variant of IGFBP-7 (IGFBP-7 v) is 165 nucleotides smaller than conventional IGFBP-7 transcripts. The deletion occurs within exon 1 but does not lead to a frameshift or an aberrant termination of the deduced protein sequence. However, the IGFBP-7 consensus motif is deleted. In contrast to non-tumorigenic granulosa cells, IGFBP-7 v is not expressed in tumor cell lines of ovarian and cervical origins. Computer based analysis of IGFBP-7 v demonstrates that the deduced amino acid sequence lacks the IGFBP typical domain [(G)CGCCXXC], which is supposed to be involved in IGF binding suggesting an IGF independent action of IGFBP-7 v.

Schlüsselwörter

Insulin - Wachstumsfaktor - Spleißen - Deletion - Tumorzelle

Key words

IGF - IGFBP - granulosa cell - splicing - deletion - tumor cell

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