Designing peptide-based FSL (Function-Spacer-Lipid) constructs
Designer red blood cells with artificial antigenic expression is created due to the fact that Standardised red cell phenotypes are not available readily. These methods include traducing cord blood cells undergoing erythroid differentiation with viral vectors or attachment of antigens onto cells using KODE technology Function Spacer Lipid (FSL). FSL constructs have been used in modifying embryos, epithelial cells, endothelial cells, RBCs,etc., They are applied to create quality control systems, modify cell adhesion/ interaction/ separation and invitro and invivo imaging of virions.
Creating a kodecyte is a simple robust process. It involves incubation of cells in lipid free media with a solution of FSL constructs for a few hours at 37o c. During the few hours of incubations the FSL constructs incorporates spontaneously with the cell membrane of the cells. Reproducibility of kodecytes is dependent on various factors like incubation time, temperature, FSL concentration and cell concentration. Consistency of the cells is achieved by maintaining these parameters at a particular ratio. FSL constructs remain with the cells for their lifetime if these cells are maintained in a lipid free media.
Theoretical and practical aspects of FSL designing
The basic structure of FSL constructs of a functional head (F), a spacer (S) and a lipid tail (L).
The choice of functional groups is mainly determined by the chemical property and solubility of those components. A large group of saccharides including blood group related determinants, sialic acids, hyaluronin polysachharides and other mioties such as biotin, flurophores, reactive functional groups (RFGs) and a range of peptides have been used as functional heads in FSL constructs.
Spacer provides construct with the property of dispersibility in water. It should also provide stability and spontaneous incorporation into the membrane. Oligomers of ethylene oxide, partially carboxyl methylated oligoglycines, shorter building blocks like diaminoethane and adipic acid residues. Applications determine the structure of a particular spacer, distance between the functional head and type of conjugation with the F. If spacer molecule is a plain peptide, highly selective thiol /maleimide ligation is employed. While terminating, peptide chain with extran cysteine molecule is employed. If a spacer is used with human serum, the spacer should be unreactive to both serum and cellular components.
The function of the lipid tail s to anchor the construct on to a surface, which can be cell membrane/ micelle or liposome. A range of lipids can be used. For instance for RBCs 1,2-dioleolyl-sn-glycero-3-phosphoethanolamine (DOPE) lipid is most suitable.
Proteins, Epitopes and Antigenic Peptides
A protein molecule is a conformational defined molecule while peptide is shorter oligopeptide chain. Both are constructed by amino acids bond by peptide bonds. Specific folding of the long chains leads to the secondary structure. Tight packing of these secondary structure leads to the tertiary structure of the protein. These tertiary structure combines with other protein molecules to form the quaternary structure. The protein molecule while binding with the antibody can undergo conformational changes. Antigens or epitopes are the part of the protein which binds with the antibody molecule. Epitopes are classified as linear/ continuous and conformational/discontinuous based on their degree of folding and continuity. Linear epitopes can be produces artificially or mimicked while conformational remains a challenge.
Mimotopes have similar physiochemical properties and spatial organization of the confromational epitope. They are selected based on their property to interact with the target molecule (antibody). Peptide mimotopes can be made for any blood type antigens. However FSL based is still a challenge.
Step 1 Antigenic peptide sequence (F) refinement in the context of FSL design
First step in construction of a FSL is to speculate the residues that re likely to be epitopes in the whole protein sequence. In case of blood groups it is easier to find as antigenic region is bond tightly. In case of organisms the first step is hard as a high number of apitopes and proteins are present. However the simplest approach of, blood group polymorphism due to variant amino acid is shown by a polymorphic epitope, is not usually applicable. It is applied only in case of linear epitopes only. The part of the peptide variant may or may not be responsible for the polymorphism. Substitution mutation of a remote amino acid may have lead conformational changes to the epitope. Fortunately glycophorins a blood group antigen is linear and can be used in the constructs of FSL.
Once the peptide sequences have been identified, they are analyzed for compatibility as an FSL construct. The compatibility is analyzed based on the following issues characteristics of proteins ⁄ peptides ⁄ amino acids can be identified by using existing algorithms ⁄ rules as found in resources such as: I-Tasser, BLAST, Immune Epitope Database, hydrophilicity, secondary structure, B-cell epitope, flexibility, surface accessibility, Nglycosylation, O-glycosylation and epitope prediction
Theoretically the length of the amino acid required for antibody recognition is 4-6. But, practically anything less than 12 is not recommended.
Thiol group present in the extra cysteine group in the spacer reacts with the carboxyl or amino end of the peptide and spacer is attached to the peptide sequence. It can be done when there is no cysteine group present in the peptide sequence. If there is cystein sequence present in the peptide sequence a substitution of cys by a-aminobutryic acid is done.
Peptides bearing N-terminal glutamine (Gln) are known to undergo notoriously fast cyclization to form pyroglutamic acid residues with the cyclization rate being in the range 2–3% ⁄ h under physiological conditions. This spontaneous transformation is likely to affect antigenicity and should be excluded where the predicted epitope begins with N-terminal Gln. This is counterd by adding an extra residue before Gln or by sacrificing the particular peptide sequence.
Peptides incorporating asparagine (Asn) followed by a non-hydrophobic residue –Asn–Xaa- are prone to spontaneous deamidation via intermediate formation of five-membered aspartimide, giving rise to a mixture of related a- and b-aspartyl peptides potentially having altered antigenic profiles. The rate of degradation depends largely upon the Xaa structure and is highest for Gly approaching ~2%⁄ h under physiological conditions. This type of intrinsic peptide instability is incurable by sequence manipulation and should be considered seriously. At least Asn–Gly should be excluded by all means while the potential problems.
The most important aspect of peptide selection is solubility. Detecting and solving the solubility problems associated with a particular peptide sequence determines the effectiveness of the construct. Ideally, to ensure smooth and efficient ligation, both should possess solubility in ligation buffer (01 M 4-methylmorpholine formate in 30% isopropanol, pH = 6.5) on the order of few mg⁄ mL. Recommended sequences marked with insolubility signatures
- Peptides either having net charge |Zpept| < 2 themselves, or yielding FSL constructs with net charge in that region, that is, |Zpept ) 5| < 2; Zpept = R(R+K+N-terminus + 0.5H) ) R(D+E+C-terminus).
- Sequences harbouring clusters formed by non-charged residues that include three or more consecutive I, F, Y, W, L, V, T.
In cases when solubility improvement through manipulation of sequence is impossible, cosolvents are used to solubilize peptide prior to ligation (i) trifluoroethanol (or hexafluoroisopropanol, or 2-methoxyethanol) – ligation buffer or pyridine (1:1, v ⁄ v); (ii) neat dimethylformamide or (iii) 6 M guanidinium hydrochloride (should be tested in this order) and may prove useful for dissolving those peptides at a concentration of few mg⁄ mL.
Dimethyl sulphoxide (DMSO) may promote disulphide formation and should not be used to dissolve Cys-containing peptides. The resultant FSL construct may be more soluble than the starting peptide and although forcing conditions (e.g. increasing pH with ammonium bicarbonate, neat DMSO and aqueous alcohols) can allow for reconstitution of poorly soluble FSL constructs, all FSL constructs we use must ultimately be dispersible in saline alone. This is a critical feature of FSLs for biological use as it allows modification of cells without affecting their vitality and functionality.
Potential glycosylation sites
Glycosylation is a common post-translational modification of eukaryotic proteins and influences folding, solubility, antigenicity, conformation, interactions and half-life of peptides. Both N- and O- potential glycosylation need to be considered during the peptide selection process. The standard N-glycosylation sequence is N-X-S ⁄ T where X could be any amino acid other than proline. There is no single O-glycosylation motif but it usually occurs with high content of serine, threonine and proline residues and in combinations like TAPP, TVXP, S ⁄ TPXP, TSAP, PSP and PST, where X is any amino acid. As glycosylation dramatically changes the nature of an epitope the following strategies should be considered: (i) optimizing the peptide to avoid residues suspected in glycosylation or (ii) direct chemical synthesis of appropriately glycosylated peptides or as a last resort (iii) using a naked peptide in the hope that it may still retain the expected binding specificity. Needless to say, these considerations also apply to any post-translational modification in the proximity of the epitope sequence.
Algorithms for comparing primary biological sequences with microbial sequences (e.g. BLAST, IEDB) help predict and evaluate the degree of potential cross-reactivity associated with the proposed peptide. Avoidance of microbial related epitopes reduces the risk of undesired nonspecific cross-reactivity from naturally occurring antibodies directed against microbes.
Transfusion of blood always associated with lot of complications, the collective term used for these complications are Transfusion-related immune modulation (TRIM). The potential of packed RBC (PRBC) to modulated recipient immune is a central area of research. FSL technology has found its application in this area. The length of ex-vivo storage of blood is studied using FSL technique. First, we tracked the binding of FSL-FLRO4 on RBC at different storage ages using a time course approach to confirm feasibility. Secondly, we further investigated differences in FSL-FLRO4 labelling during RBC storage by labelling isolated light-young- and dense-old-RBC from several individual PRBC units.
Material and Methods
FSL-FLRO4 constructs were obtained from Kode Biotech Materials Limited. The construct is comprised of fluorescein (FLRO4) linked to an activated adipate derivative of dioleoyl phosphatidyl ethanolamine. Phosphate buffered saline was used to reconstitute lyophilized FSL-FLRO4 (stock concentration 2 mg/mL), prepare FSL-FLRO4 working concentration (50 μg/mL) and as a wash buffer following RBC labelling.
PRBC units obtained one day after standard processing and filtration procedures. RBC from a leuko depleted PRBC unit were labelled (see below) and assessed using a time course approach (n= 6 independent experiments). To further assess the differences in the capacity of FSL-FLRO4 labelling, the PRBC unit was centrifuged to separate “young” and “old” RBC (n= 4 independent experiments). Based on the differential separation of young and old RBC reported by Sparrow et al. the light-young- (top 10% of RBC layer) and dense-old-RBC (bottom 10% of RBC layer) were obtained via density distribution centrifugation (3220 g, 30 mins at 4 0C) in 50mL Falcon tubes .
FSL-FLRO4 labelling of RBC
100μL of FSL-FLRO4 (50 μg/mL in PBS) was added to an equal volume of RBC and mixed by vortexing. Cells were incubated at 37oC for one hour, followed by three washes with PBS and centrifugation (515 g, 5 mins at room temperature). In parallel, a matched RBC control was prepared with the omission of FSL-FLRO4 in PBS. Both FSL-FLRO4 and control RBC were stored at 4 ± 2 °C before flow cytometric assessment at the defined storage age (D2, D7, D14, D21, D28, D35, D42).
At each time point, 3 μL of FSL-FLRO4 labelled RBC or unlabelled RBC control were assessed using FACSCanto II flow cytometer. Fluorescein emission was collected with 530/30 band pass filter following excitation with 488 nm laser. Unlabelled RBC controls were used at each time point to establish the quadrants for FSL-FLRO4+ RBC.
FSL-FLRO4 label was retained by PRBC for the duration of routine storage Substantial biochemical and biomechanical changes occur during routine storage of PRBC. To facilitate tracking and interaction of stored PRBC in both in-vitro and in-vivo transfusion models, a reliable method of RBC labelling is required. We assessed whether FSL-FLRO4 would be retained by PRBC during routine storage. 100% of labelled RBC remained positive for FSL-FLRO4 during routine PRBC storage. Over the time course, a significant decrease (P<0.0001) in the intensity of FLRO4 was observed. In comparison to the FSL-FLRO4 MFI at D2, an average reduction of 66% was observed by the date of PRBC expiry, D42 (P<0.001). This reduction was particularly apparent from RBC at D35 (P<0.0001) onwards. Despite this reduction over time, all cells were clearly identified as FSL-FLRO4+ following storage for 42 days. Together these data demonstrate FSL-FLRO4 is a suitable reagent for labelling RBC during storage of PRBC for visualization and tracking.
Age of RBC did not impact uptake of FSL-FLRO4 into the cell membrane
Mature enucleated RBC are constantly renewed from nucleated precursors in the bone marrow. Human RBC have an average life span of 120 days in circulation and a RBC donation for transfusion contains a heterogeneous population of both recently enucleated (light-young) and older RBC (up to 120 days, dense-old). Given the decrease in FSL-FLRO4 intensity on labelled RBC overtime, we considered that FSL-FLRO4 may remain more stably inserted in the membrane of younger RBC, and the decrease in intensity of FSL-FLRO4 observed during storage may be due to reduced labelling of older RBC. FSLFLRO4 inserted into both light-young- and dense-old-RBC, and both subsets remained FSLFLRO4 + for the duration of the time course. Assessment of mean MFI indicated that there was no difference in the capacity of light-young- and dense-old-RBC to take up the label, and a similar loss of intensity was observed during storage.
FSLFLRO4 uptake and release do not change for both light/young or dense old RBCs. The uptake and release of RBC does not differ any phase of RBC storage. Thus FLSFLRO4 is a reliable technology for labelling RBCs irrespective of their age. This technique can be used for visualization and tracking red blood cells during storage.