Despite several recent studies, little information is available at the molecular level on the molecular factors and mechanisms involved in the secretory pathway of insect cells in general, and the lepidopterian Sf9 cells in particular [46
]. Secreted recombinant proteins are usually obtained by fusion of the protein of interest to the N-terminal leader peptide of the honeybee pro-mellitin (MBM-SP) [47
]. However, this strategy does not always guarantee the secretion of the recombinant protein. For example, scSCR20, a MBM-SP-leaded scFv derived from an anti-African cassava mosaic virus monoclonal antibody [49
] was not secreted in the culture medium of BV-infected insect cell lines from Spodoptera frugiperda
, Trichoplusia ni
, and Mamestra brassicae
. However, scSCR20 molecules were released at high levels in the culture medum of Drosophila
cell lines stably expressing MBM-SP-fused scSCR20 [50
]. Likewise, certain types of scFv expressed in insect cells using recombinant BV have been recovered in the extracellular medium although they lack an insect cell leader peptide [51
]. The most probable explanation was that specific features of recombinant scFv, such as their own N-terminal amino acid sequence or/and other downstream domain(s), can influence their behaviour in BV-infected cells.
BV-display has been used for almost a decade for immunisation purposes, gene delivery, or development of eukaryotic libraries [14
]. Conventional BV-display involves baculoviral envelope glycoprotein GP64 manipulations [17
], or the use of the VSV-G stem [16
]. This differs from the incorporation of foreign proteins or glycoproteins into the baculoviral envelope without fusion to GP64, such as the envelope incorporation and display of functional human beta-2 adrenergic receptor (ß2AR) described in an earlier study [54
]. In a more recent work, we demonstrated the incorporation of the human CAR glycoprotein into the baculoviral envelope. CAR is the high affinity receptor for adenovirus serotype 5 (Ad5) and is a resident glycoprotein of the human cell plasma membrane. The baculoviral envelope-incorporated CAR was fully functional at the surface of BVCAR
virions, and enabled the formation of BVCAR
-Ad5 complexes, mediated by the interaction between the adenoviral fiber and CAR. We have used this strategy of BVCAR
-Ad5 duo formation to transduce Ad5-refactory cells [41
It was relatively easy to conceive that human ß2AR and CAR molecules, even though expressed in heterologous system, could be displayed on the baculoviral envelope since both are resident membrane glycoproteins. It was rather unexpected for scFvE2/p17, which was an artificial molecule extrinsic to the BV-insect cell system. Moreover, in the case of scFvE2/p17, the scFv molecule was not constructed for membrane targeting, in contrast to scFvG2/p17 which carried the specific Met-Gly dipeptide signal for N-myristoylation by N-myristoyl-transferases. Comparison of the amino acid sequences of scFvE2/p17 and scFvG2/p17, which both lacked any consensus leader peptide, showed that they only differed by three residues at their N-terminus, M(EAS)L for scFvE2/p17, versus M(G)L for scFvG2/p17. The results of these minor sequence changes were drastic in terms of scFv solubility, cell compartmentalization and extracellular release. Recombinant scFvG2/p17 protein expressed in Sf9 cells was inexploitable since it was insoluble and trapped in the membrane pellet.
Recombinant scFvE2/p17 however was recovered simultaneously under two different forms: (i) as soluble scFv molecules from lysates of BV-infected Sf9 cells, and (ii) as BV-displayed scFv in the culture medium of the BV-infected Sf9 cell cultures. Both forms could be used as biological tools for different purposes. Soluble scFvE2/p17 could serve in conventional diagnostic assays for HIV-1 Gag detection, through specific recognition of the conserved MAp17 epitope. MAp17 functions as a structural component of HIV-1 virions, but also as a viral cytokine which binds to a cellular receptor, p17R [55
], when released by HIV-infected cells. In the case of BV-displayed scFvE2/p17, the potential applications would be different. For example, if one considers the virokine properties of soluble MAp17 and the importance of inflammatory response at the mucosal sites of HIV-1 entry [55
], one could envisage to use pelletable, BV-displayed scFvE2/p17 in experimental models of infected mucosae to deplete soluble MAp17 from the extracellular medium, or/and to compete with MAp17 for binding to p17R.
To assess the role of the N-terminal domain of scFvE2/p17 in the process of membrane addressing and scFv display on the baculoviral envelope, we fused the N-terminal octadecapeptide 1
(abbreviated N18E2) to another bioactive scFv molecule, scFv-M6-1B9. The ligand of scFv-M6-1B9 is M6, also called CD147 [38
], a transmembrane glycoprotein highly expressed in various types of malignant cells [57
] and tumors, e.g. nasopharyngeal carcinoma [58
]. CD147 acts as an inducer of extracellular matrix metalloproteinases (EMMPRIN is another acronym for CD147) to promote tumor growth, invasion, metastasis and neoangiogenesis, and is a prognostic marker for invasiveness in prostate cancer [59
] and thyroid carcinoma [60
]. CD147 is also involved in atherosclerosis plaque instability [61
] and in the regulatory inhibition of starvation-induced autophagy in human hepatoma cells [62
We expressed the chimeric scFv-N18E2/M6 molecule in recombinant BV-infected Sf9 cells, and found that the BV progeny displayed scFv-N18E2/M6 on the baculoviral envelope. This suggested that the N-terminal octadecapeptide N18E2 carried the function required for BV-display of scFv molecules, and could be considered as a BV envelope addressing/anchoring signal peptide. This was further supported by the comparison of the structural domains of scFv downstream of the N18E2 peptide: scFv-N18E2/M6 and scFvE2/p17 differed by the successive order of their variable regions, VL-linker-VH from the N- to C-terminus in scFv-M6-1B9, versus VH-linker-VL in scFvE2/p17. This implied that the nature of the variable region downstream of N18E2 had little influence, if any, on the membrane addressing of chimeric, N18E2-fused scFv.
Although the molecular mechanism of cell trafficking of our chimeric scFv-N18E2/M6 molecule still remained to be elucidated in molecular terms, our present data provided a novel concept and platform for engineering scFv molecules competent for BV-display.