Characterisation of antibodies to Migration Stimulating Factor (MSF). Detection of MSF isoforms.

Migration Stimulating Factor (MSF) is a 70kDa truncated isoform of fibronectin (FN). Unlike FN, MSF is not a matrix molecule but a soluble factor which exhibits a range of potent cytokine-like bioactivities not displayed by full-length FN. Two isoforms of human MSF (MSF+aa and MSF-aa), as well as murine Migration Stimulating Factor (mMSF) have been cloned. In this communication we report the characterisation of various polyclonal and monoclonal antibodies to human and murine MSF. In particular:

been cloned. In this communication we report the characterisation of various polyclonal and monoclonal antibodies to human and murine MSF. In particular: Specific MSF-identification antibodies that recognise both MSF+aa and MSF-aa; (ii) Specific mMSF-identification antibodies; (iii) Identification antibodies that recognise MSF-aa but not MSF+aa; (iv) MSF-function-neutralising antibodies that recognise MSF+aa, MSF-aa, mMSF and the gelatin-binding domain of FN/MSF (Gel-BD) but not full-length FN.

Description of the antigens:
Fibronectin (FN) is a modular glycoprotein consisting of the following functional domains: Hep 1/Fib-1 (N-terminal low affinity binding to heparin and fibrin), Gel-BD (binding to gelatin/collagen), Cell-BD (RGD-mediated binding to integrins), Hep-2 (high affinity heparin binding) and Fib-2 (C-terminal fibrin binding site). Each functional domain is composed of a different number of three possible homology modules, called type I, II and III (Fig 1).
Human Migration Stimulating Factor (referred to as MSF) is a 70kDa truncated isoform of FN. MSF RNA is generated from the fibronectin gene by a two-stage processing mechanism.
In the first stage, an MSF-specific primary transcript is generated from the fibronectin gene by read-through of intron 12, separating exons III-1a and III-1b. This is followed by intraintronic cleavage to produce a 5.9 kb MSF pre-message that remains sequestered within the nucleus, where it is rapidly degraded. In cells that express MSF protein a second stage takes place, whereby the intron-derived 3' UTR of the pre-message is cleaved a second time to produce a 2.1 kb mature MSF message. This has a shorter (195bp) intron-derived 3' sequence containing a 30bp in-frame coding sequence (immediately contiguous with exon III-1a), followed by a 165bp 3'-UTR containing several in-frame stop codons and a cleavage/polyadenylation signal. The mature message is rapidly exported to the cytoplasm for translation [Schor et al, 2003;Kay et al, 2005]. Therefore, MSF is identical to the N terminus of full-length fibronectin, up to and including the amino acid sequence coded by exon III-1a, with the addition of an MSF-unique (intron-coded) 10 amino acid C-terminus: VSIPPRNLGY [Schor et al, 2003;Kay et al, 2005] (Fig 1).
FN is a major component of the extracellular matrix. Unlike FN, MSF is a monomer and is not a matrix molecule but a soluble factor which exhibits a range of cytokine-like bioactivities, including the stimulation of cell migration and angiogenesis. The motogenic activity of MSF is mediated by the IGD motifs, present in modules I3, I5, I7 and I9 of the Gel-BD domain and, in some cases, by the HEEGH motif present in module I8 [Schor et al, 1999[Schor et al, , 2003Houard et al, 2005;Millard et al, 2007] (Fig 1, Fig 2). Two isoforms of MSF (referred to as MSF) have been cloned. Both contain the same unique 10 amino acid C-terminus as well as same bioactive IGD and HEEGH amino acid motifs.
The two isoforms differ solely in terms of a 45bp deletion in exon II-1 and are consequently referred to as MSF+aa and MSF-aa to indicate the retention or deletion of a 15 amino acid sequence in module II-1 (Fig 1 and Fig 2). The term MSF or total MSF will be employed to denote both isoforms.
We have isolated and cloned a murine MSF (mMSF) transcript by PCR. This is homologous to its human counterpart, consisting of the 5-terminus of mouse FN, up to and including exon III-1a, and terminating in a unique 3'coding sequence derived from the intron separating exons III-1a and -1b. The 3'UTR ends in a polyA tail. mMSF protein consequently has a molecular mass of 70kDa and terminates in a unique 12 amino acid C-terminus: VSNSSAALDSDP (Fig 3). The murine FN coding sequence is over 90% homologous to human FN; the four IGD motifs and the HEEGH motif are similarly located in modules I3, I5, I7, I9 and I8, respectively. However, there is no significant homology between the human and mouse intron-derived C-terminal MSF-unique peptides.
Eukaryotic and prokaryotic recombinant MSF and mMSF were produced as described [Schor et al 2003].
We have raised polyclonal (Pab) and monoclonal (Mab) antibodies to human and murine MSF. The peptides used as antigens to raise these antibodies and an overview of the results obtained are shown in Table 1. The antibodies were characterised by ELISA, immunoblotting, IHC and their ability to abrogate or remove MSF/mMSF bioactivity [Schor et al 2003[Schor et al , 2012. As expected, different antibodies were useful for certain techniques and not for others.

Production of antibodies
To be completed

Characterisation of VSI antibodies
To be completed Conclusions: VSI are MSF-specific identification antibodies that recognise the unique 10-mer C-terminal sequence of MSF+aa and MSF-aa; that is, total MSF.

Characterisation of TYN antibodies
To be completed Conclusions: TYN are identification antibodies that recognise MSF-aa but not MSF+aa.

Characterisation of VSN antibodies
To be completed Conclusions: VSN are mMSF-specific identification antibodies that recognise the unique 12mer C-terminal sequence of mMSF.

Characterisation of pepQ antibodies
To be completed Conclusions: pepQ are MSF-function-neutralising antibodies that recognise MSF+aa, MSFaa, mMSF and Gel-BD but not FN.

Methods
To be completed  I  I  I  I  I I  I  I I  II  II   III-1a   MSF+aa   Gel-BD   I  I  I  I  II  I I I  IIICS  I  I  I  III  III III  III  III  III  III  III  III  III  III   Hep 1/Fib-1 (N-terminal low affinity binding to heparin and fibrin), Gel-BD (binding to gelatin/collagen), Cell-BD (RGD-mediated binding to integrins), Hep-2 (high affinity heparin binding) and Fib-2 (C-terminal fibrin binding site). Each domain is composed of three possible homology modules, called type I, II and III. MSF is identical to the N terminus of fibronectin, up to and including the amino acid sequence coded by exon III-1a, with the addition of an MSF-unique (introncoded) 10 amino acid C-terminus. Two isoforms of MSF have been cloned. These differ solely in terms of a 45bp deletion in exon II-1 and are consequently referred to as MSF+aa and MSF-aa to indicate the retention or deletion of a 15 amino acid sequence in module II-1. The location of IGD motifs (↓) and HEEGH motif (*) is indicated.