3 Results
3.3 Sema3a is expressed, processed and secreted by O9-1 cells
isoforms
Related class 3 semaphorins are described to exist in monomeric, dimeric and processed isoforms (Adams et al., 1997; Christensen et al., 2005). A schematic representation of the full-length Sema3a protein composition with the predicted processing sites and the potential isoforms, resulting from proteolytic processing, is illustrated in Figure 12.
Figure 12: Schematic representation of full-length Sema3a protein. Positions of the signal sequence (red), the Sema (Semaphorin) domain (blue), the PSI (plexin-semaphorin-integrin) domain (yellow), the Ig (immunoglobulin)-like domain (green), the basic domain (purple) and the predicted processing sites 1, 2 and 3 (black arrowheads) are indicated. Sema3a sequences containing the processing sites are represented within the figure (top). Processing site after signal sequence (open arrowhead) is permanently used. Theoretical molecular weights of cleavage products are shown in kilo Dalton (kDa). Ab: binding region of the used anti-Sema3a antibody.
Adapted from Adams et al. (1997) and Christensen et al. (2005).
After Sema3a expression was identified in O9-1 cells by RT-PCR analysis (chapter 3.2, Figure 10), the expression of Sema3a was investigated on protein level using cytoplasmic and nuclear O9-1 protein extracts. Secreted Sema3a was analyzed using concentrated
cell culture medium of O9-1 cells. The cytoplasmic and nuclear protein extracts were separately isolated and the medium was concentrated after 24 h of usage. In order to check monomeric and dimeric Sema3a protein isoforms, the different proteins were treated with the reducing agent DTT (+DTT) or without reducing agent (−DTT). Medium and protein extracts were analyzed by western blot analysis using an anti-Sema3a antibody and 30 µg of protein per lane.
The representative results are shown in Figure 13. The monomeric isoforms of ~87 kDa (p87), ~85 kDa (p85), ~82 kDa (p82) and ~61 kDa (p61) were observed in concentrated medium with reduction, whereas the dimeric isoforms of ~174 kDa (p87:p87) and
~170 kDa (p85:p85) as well as the monomeric isoforms of ~82 kDa (p82) and ~61 kDa (p61) were detected in concentrated medium without reduction. In cytoplasmic extract with reducing agent, the dimeric isoform of ~170 kDa (p85:p85) and the monomeric isoform of
~85 kDa (p85) were observed. The dimeric isoform p85:p85 possibly indicates an incomplete cleavage of the disulfide bond between the dimer although the reducing agent DTT was added. In cytoplasmic extract without reducing agent, the dimeric isoform of
~170 kDa (p85:p85) was received. The monomeric isoform of ~85 kDa (p85) was observed in nuclear extract with reduction, whereas the dimeric isoform of ~170 kDa (p85:p85) was received without reduction. In nuclear extract, with and without addition of reducing agent, potentially unspecific protein bands due to unspecific binding of the used antibody were detected. To proof this assumption, O9-1 cells with a full-lengths Sema3a plasmid containing a HA-tag between the signal sequence and the semaphorin domain should be performed. By detection the tag using an anti-HA antibody, the same protein band pattern without the potentially unspecific bands should be received.
Detection of α-Tubulin (~50 kDa protein band) served as loading control for the cytoplasmic extracts. However, the detection of α-Tubulin in the nuclear extracts revealed the additional existence of cytoplasmic proteins. This fact can be possibly explained by the used protein extraction method. The nuclear protein Baf47 (~47 kDa protein band) was solely received in the nuclear extracts.
Collectively, the investigation of Sema3a in O9-1 cells on protein level revealed that the expressed full-length Sema3a protein undergoes proteolytic processing to shorter isoforms. Moreover, the existence of monomeric and dimeric isoforms as well as the secretion of Sema3a was demonstrated. The obtained results are summarized in Table 1 and Figure 14.
Figure 13: Isoforms of Sema3a resulting from proteolytic processing and dimerization.
Cell culture medium was concentrated (cM) after 24 h of usage. Cytoplasmic (C) and nuclear (N) protein extracts of O9-1 cells were separately isolated. 30 µg of protein treated with the reducing agent DTT (+DTT) or without reducing agent (−DTT) was used per lane. The Sema3a isoforms were analyzed by western blot analysis using an anti-Sema3a antibody. In concentrated medium with reducing agent the monomeric isoforms of ~87 kDa (p87), ~85 kDa (p85), ~82 kDa (p82) and
~61 kDa (p61) were detected, whereas in concentrated medium without reducing agent the dimeric isoforms of ~174 kDa (p87:p87) and ~170 kDa (p85:p85) and the monomeric isoforms of ~82 kDa (p82) and ~61 kDa (p61) were observed. In cytoplasmic extract with reduction, the dimeric isoform of ~170 kDa (p85:p85) and the monomeric isoform of ~85 kDa (p85) were received. The dimeric isoform p85:p85 possibly indicates an incomplete cleavage of the disulfide bond between the two p85 monomeric isoforms despite DTT addition. Without reduction, solely the dimeric isoform of
~170 kDa (p85:p85) was observed. In the nuclear extract with reducing agent, the monomeric isoform of ~85 kDa (p85) was received, whereas without reducing agent the dimeric isoform of
~170 kDa (p85:p85) was observed. The detection of α-Tubulin (~50 kDa protein band) served as loading control for the cytoplasmic extracts, revealing the additional existence of cytoplasmic proteins also in the nuclear extracts which can be possible traced back to the used protein extraction method. The nuclear protein Baf47 (~47 kDa protein band) was exclusively detected in the nuclear extracts. Stars () illustrate potentially unspecific protein bands due to unspecific binding of the anti-Sema3a antibody. Molecular weights of proteins and marker bands are indicated in kilo Dalton (kDa).
Table 1: Sema3a isoforms detected in concentrated medium as well as cytoplasmic and nuclear protein extracts of O9-1 cells. Medium and protein extracts were treated with the reducing agent DTT (+DTT) and without reducing agent (−DTT) and analyzed by western blot analysis usingan anti-Sema3aantibody. Approximate molecular weights of the isoforms are given in kilo Dalton (kDa).
concentrated medium cytoplasmic extract nuclear extract
+DTT −DTT +DTT −DTT +DTT −DTT
87 kDa 174 kDa 170 kDa 170 kDa 85 kDa 170 kDa
85 kDa 170 kDa 85 kDa
82 kDa 82 kDa 61 kDa 61 kDa
Figure 14: Schematic representation of proteolytic processed and dimerized Sema3a isoforms identified in concentrated medium as well as cytoplasmic and nuclear protein extracts of O9-1 cells. Isoforms resulting from combined proteolytic processing and dimerization of Sema3a full length protein are shown, which were detectable with the used anti-Sema3a antibody by western blot analysis. (A) Four monomeric isoforms were observed: ~87 kDa (p87),
~85 kDa (p85), ~82 kDa (p82) and ~61 kDa (p61). (B) Two dimeric isoforms were detected: the dimeric isoform of ~174 kDa (p87:p87) consist of two ~87 kDa (p87) monomeric isoforms, whereas the dimeric isoform of ~170 kDa (p85:p85) consist of two ~85 kDa (p85) monomeric isoforms.
Disulfide bonds between the dimeric isoforms are indicated with −S−S−. Approximate molecular weights of the isoforms are displayed in kilo Dalton (kDa) and in short version (p) (bottom).
Adapted from Adams et al. (1997) and Christensen et al. (2005).