Through COS7 cell surface expression and rosetting adhesion assays, this study has identified an erythrocyte-binding domain within the N-terminus of one of two P. knowlesi RBL proteins, namely PkNBPXb. This binding domain wase called PkNBPXb-II, because it was the second in a series of eight overlapping protein constructs tested. Using similar procedures, a comparable domain could not be identified near the N-terminus of PkNBPXa. Importantly, however, the only other RBL ligand expressed by P. knowlesi merozoites, PkNBPXa, in contrast with PkNBPXb, strongly binds human erythrocytes in addition to monkey host erythrocytes by traditional EBAs.
This study focused on the N-terminal half of the PkNBPs  given certain intuitive considerations about RBL structure and the exposure of binding sites, as well as previous studies characterizing the binding domain of PvRBP1 . In addition, erythrocyte-binding domains of P. falciparum and P. yoelii RBL family members have since been identified in various N-terminally associated regions [22, 23, 33].
PkNBPXb-II was the only domain found to bind erythrocytes. This domain includes amino acids 184 to 531, contains five cysteines, and resides in the relatively cysteine-rich N-terminal area designated as the Rh homology region in RBL paralogs in P. falciparum[23, 25, 34]. Interestingly, this region of low homology was first delineated by clustal alignment of P. vivax RBP1 with Rh1 and Rh4 . The binding domains for PfRH4 and PfRH5 appear to reside in this zone of weak homology based on the binding of recombinant peptides designed from this region [23, 25]. However, the binding domains for two other P. falciparum family members, Rh1 and Rh2a/Rh2b, apparently reside just outside this region of Rh homology [22, 24, 26].
The RBL invasion ligand proteins contain a variable number of cysteines in the N-terminal region of homology, but it has not been known if these residues participate in disulphide bond conformation necessary for receptor-ligand binding interactions. This study shows that the mutation of four individual cysteine residues prevented the trafficking to and expression of the PkNBPXb-II protein on the surface of COS7 cells, suggesting that cell-surface expression of PkNBPXb-II is dependent on critical cysteine residues. Only the mutation of Cys193 to Gly did not completely abolish the binding of NBPXb-II to erythrocytes, but expression was reduced along with a reduction of binding levels by 85 or 90 % as compared to cells expressing unaltered NBPXb-II. Taken together the data suggest that cell-surface expression of PkNBPXb-II is dependent on critical cysteine residues and this lack of trafficking to the surface could indicate that disulphide formation is a functionally important feature in this region of the protein. Mutagenesis of amino acid residues has been performed to map Plasmodium blood-stage parasite binding domains in PfEMP1DBLβ-C2  and PvDBP [36, 37]. Although functionally important cysteine-rich regions have been predicted within the N-terminal regions of PfRH1, PfRH2a/2b, PfRH4 and Py235 [23, 24, 33, 38, 39], there have been no studies conducted, with mutations or otherwise, to show cysteine functionality in these ligands. The RBL N-terminal regions share at least three conserved cysteines (Figure 3), and based on this data it is reasonable to hypothesize that they may be important for the conformational dependent functions of some or all of the RBLs.
Different enzymatic treatments of erythrocyte target cells can be useful to generate receptor profiles and delineate potential receptors for particular binding proteins. This study shows that chymotrypsin treatment of rhesus erythrocytes abolished the formation of erythrocyte rosettes with PkNBPXb-II, but not trypsin or neuraminidase treatments. In contrast, adhesion of native PkNBPXb to treated or untreated rhesus erythrocytes in EBAs was positive, although binding was consistently stronger with neuraminidase-treated or untreated cells. These data indicate that PkNBPXb-II contains a binding domain when tested in rosetting assays, yet the native protein may have additional co-functional domains resistant to enzyme treatment. Recently, ligand-binding specificity has been studied in detail, including enzymatic cleavage profiles in PfRh2a/b a homolog of the PkNBPs that suggest more than one binding domain in a RBL invasion ligand .
Native PkNBPXb as well as the PkNBPXb-II domain also has a host specific target cell binding preference. Native PkNBPXb in EBAs bound to rhesus monkey erythrocytes, but not to human erythrocytes, a result similar to that of rosetting assays with PkNBPXb-II. PkNBPXb-II robustly bound rhesus erythrocytes and even better when the rosetting erythrocytes were from the primary monkey host, M. fascicularis or mangabey and gibbon species. However, this binding domain and native PkNBPXb did not bind erythrocytes from humans, chimpanzees and for the most part New World Monkeys.
Plasmodium knowlesi has emerged as an important zoonotic human pathogen of increasing public health significance [1, 2, 5, 41]. A future focus on studying PkNBPXa adhesion will be important because, in contrast to PkNBPXb, PkNBPXa not only binds to rhesus erythrocytes in EBA assays , but also human erythrocytes (Figure 5). This study attempted to identify a binding domain in PkNBPXa. However, while seven segments of PkNBPXb were readily cloned and expressed at the surface of COS7 cells, comparable cloned segments of PkNBPXa that are surface expressed have not yet been developed, despite many attempts. Future attempts to optimize both expression and trafficking of PkNBPXa regions may include the adjustment of the boundaries of these regions or changes in the expression vector, mammalian host cells, or expression conditions. Expanded studies involving PkNBPXa adhesion to human and other non-human primate erythrocytes will hopefully elucidate the role of this protein, perhaps as the RBL ligand used by P. knowlesi to naturally infect humans.
Interestingly, the host origin of erythrocytes correlates to some degree with the binding specificities observed, regardless of the actual ability of P. knowlesi to invade a particular primate cell type. For example, PkNBPXb binds strongly to erythrocytes from Old World monkeys (long-tailed, rhesus and pigtail macaques) with a Southeast Asian or African origin (sooty mangabeys) and to Lesser Apes (gibbons), which also originate in Southeast Asia, but not to erythrocytes from humans (or chimpanzees) or New World primates. Rosetting of macaque erythrocytes was expected because rhesus macaques are utilized as an experimental model studying P. knowlesi infections, and pigtail and long-tailed macaques are the natural hosts for P. knowlesi. Less expected, because human and chimp erythrocytes did not bind, was the strong binding observed to gibbon erythrocytes. Gibbons are neither natural nor normally experimental hosts for P. knowlesi, but can be infected with this parasite. Although New World monkeys, squirrel, owl and marmoset monkeys are known to be very susceptible to P. knowlesi infection [42–44], erythrocytes from these species did not form rosettes with PkNBPXb-II. Similarly, and importantly, human erythrocytes did not bind PkNBPXb-II or native NBPXb, even though P. knowlesi does infect humans. Since PkNBPXa not only binds erythrocytes from Old World monkeys, but also erythrocytes from humans, this RBL ligand may play an important role in allowing P. knowlesi to infect this host. These observations are consistent with the necessity of multiple receptor-ligand interactions being important to achieve successful invasion of erythrocytes from a wide range of hosts.