Belur N. Manjula

Sequence homology of group a streptococcal Pep M5 protein with other coiled-coll proteins

Group A streptococcal Pep M5 protein, an antiphagocytic determinant of the bacteria, is an alpha-helical coiled-coil molecule, and exhibits significant sequence homology with tropomyosin and myosin, but to a lesser degree with other coiled-coil proteins. Moreover, Pep M5 is more homologous to myosin than to tropomyosin, and the homologies are more numerous between the C-terminal domain of the Pep M5 protein and the S2 fragment of myosin. The C-terminal domain of the Pep M5 protein exhibits extensive sequence identity with the C-terminal region of Pep M6 molecule, another M protein serotype. Thus, regions within two M protein serotypes are homologous to the S2 region of the myosin molecule. These observations are consistent with the immunological findings of other investigators and thus may explain some of the previously reported immunological cross-reactions between antigens of the group A streptococcus and mammalian heart tissue.

Application of reductive dihydroxypropylation of amino groups of proteins in primary structural studies: identification of phenylthiohydantoin derivative of ε-dihydroxypropyl-lysine residues by high-performance liquid chromatography

The general utility of reductive alkylation of amino groups of proteins with glyceraldehyde (2,3-dihydroxypropionaldehyde) in the presence of sodium cyanoborohydride, i.e. dihydroxypropylation, as an aid in generating arginine peptides of proteins by tryptic digestion has been investigated. The dihydroxypropylation of the amino groups of ribonuclease A and the streptococcal Pep M5 protein proceeds predominantly to the stage of monoalkylation. The derivatized lysine namely, epsilon-dihydroxypropyl-lysine is stable to acid hydrolysis, and is eluted slightly ahead of histidine in the amino acid analyzer. The peptide bonds of epsilon-dihydroxypropyl-lysine residues are resistant to tryptic digestion. The arginine peptides of dihydroxypropylated ribonuclease A, and dihydroxypropylated streptococcal Pep M5 protein have been isolated by reversed-phase high-performance liquid chromatography (HPLC) of the tryptic digest of the derivatized proteins. The phenylthiohydantoin (PTH) derivative of epsilon-dihydroxypropyl-lysine has been prepared. It is eluted at a position intermediate to that of the PTH derivatives of proline and tryptophan in reversed-phase HPLC on DuPont Zorbax ODS columns. Thus the PTH-epsilon-dihydroxypropyl-lysine could be identified during the sequence studies of the dihydroxypropylated peptides. The presence of dihydroxypropyl groups on the epsilon-amino groups of lysine residues in the dihydroxypropylated peptides does not interfere with the Edman degradation studies. The ease of the dihydroxypropylation reaction, the resistance of the peptide bonds of epsilon-dihydroxypropyl-lysine residues to trypsin, and the identification of the PTH derivative of epsilon-dihydroxypropyl-lysine residues by reversed-phase HPLC makes the dihydroxypropylation procedure a valuable addition to the arsenal of procedures for limiting the tryptic digestion to the arginine residues of proteins and peptides.

Relation of streptococcal M protein with human and rabbit tropomyosin: The complete amino acid sequence of human cardiac alpha tropomyosin, a highly conserved contractile protein

Partial sequences of group A streptococcal M proteins exhibit up to 50% sequence identity with segments of rabbit skeletal tropomyosin. It is well recognized that rheumatic fever and rheumatic heart disease in humans are sequelae of group A streptococcal infection. To examine whether the human cardiac tropomyosin would exhibit greater homology with the streptococcal M proteins, we have now determined its complete amino acid sequence. The amino acid sequence of human cardiac tropomyosin was established from sequence analyses of its peptides derived by enzymic and chemical cleavages, and comparison of these sequences to the reported sequence of rabbit skeletal tropomyosin. These studies have revealed that the amino acid sequence of human cardiac alpha tropomyosin is identical to that of the rabbit skeletal alpha tropomyosin, but for a single conservative substitution of Arg/Lys at position 220. This observation increases the significance of the previously observed sequence homology between streptococcal M protein and rabbit skeletal tropomyosin and may have relevance to the pathogenesis of rheumatic fever. Furthermore, these results rank tropomyosin as one of the most highly conserved contractile proteins between vertebrate species reported thus far.

Molecular aspects of the phagocytosis resistance of group a streptococci

Once the streptococci are ingested by the phagocytic cells, they are almost all killed within minutes (93). Thus, the invasiveness is dependent upon factors which impede the phagocytosis of the bacteria. Streptococci have long been recognized to have two such antiphagocytic factors namely, the hyaluronic acid capsule, and the M protein, a component of the bacterial cell wall. The hyaluronic acid capsule is often shed spontaneously from the organism (19, 51, 58, 61), and is also destroyed readily by factors present in human serum. Therefore, the major antiphagocytic determinant of the group A streptococcus is the M protein.

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.

The amino-terminal region of group A streptococcal M protein determines its molecular state of assembly and function.

Group A streptococcal M protein, a major virulence factor, is an alpha-helical coiled-coil dimer on the surface of the bacteria. Limited proteolysis of type 57 streptococcus with pepsin released two fragments of the M57 molecule, with apparent molecular weights of 32,000 and 27,000 on SDS-PAGE. However, on gel filtration under nondenaturing conditions, each of these proteins eluted as two distinct molecular forms. The two forms corresponded to their dimeric and monomeric state as compared to the gel filtration characteristics of known dimeric coiled-coil proteins. The results of sedimentation equilibrium measurements were consistent with this, but further indicated that the “dimeric form” consisted of a dimer in rapid equilibrium with its monomer, whereas the “monomeric form” does not dimerize. The monomeric form was the predominant species for the 27 kD species, whereas the dimeric form predominated for the 32 kD species. Sequence analysis revealed the 27 kD species to be a truncated derivative of the 32 kD PepM57 species, lacking the N-terminal nonheptad region of the M57 molecule. These data strongly suggested that the N-terminal nonheptad region of PepM57 is important in determining the molecular state of the molecule. Consistent with this, PepM49, another nephritis-associated serotype, which lacks the nonheptad N-terminal region, also eluted as a monomer on gel filtration under nondenaturing conditions. Furthermore, removal of the N-terminal nonheptad segment of the dimeric PepM6 protein converted it into a monomeric form. The dimeric molecular form of both the 32 kD PepM57 and the 27 kD PepM57 did not represent a stable state of assembly, and were susceptible to conversion to the corresponding monomeric molecular forms by simple treatments, such as lyophilization. The 27 kD PepM57 exhibited a greater propensity than the 32 kD species to exist in the monomeric form. The 32 kD species contained the opsonic epitope of the M57 molecule, whereas the 27 kD species lacked the same. This is consistent with the previous reports on the importance of the N-terminal region of M protein for its opsonic activity. Together, these results strongly suggest that, in addition to its importance for the biological function, the N-terminal region of the M protein plays a dominant role in determining the molecular state of the M molecule, as well as its stability.

Domain structure and molecular flexibility of streptococcal M proteinIn Situ probed by limited proteolysis

Serologically distinct group A streptococcal M proteins, the antiphagocytic determinants of the bacteria, have a highly repetitive sequence and exhibit a heptad periodicity characteristic of alpha-helical coiled-coil proteins. Based on the differences in the pattern of heptad periodicity, the coiled-coil region of the complete M molecule has been divided into three distinct domains: I, II, and III. Domains I and II together constitute the variable part of M protein, whereas domain III is conserved among serotypes. Pepsin treatment of the M5, M6, and M24 streptococci results in a preferential cleavage of their M molecules between the predicted domains II and III, releasing biologically active fragments of the respective M proteins. Thus, a pepsin cleavage site at the junction of their variable and conserved regions is conserved in the M5, M6, and M24 proteins. In contrast, in the case of the M49 streptococci, the primary site of pepsin cleavage was observed to be within the conserved region of the M49 molecule, rather than at the junction of its variable and conserved regions. Despite containing part of the conserved region, the PepM49 protein is significantly smaller than the pepsin fragments of the M5, M6, and M24 proteins, which contain only the variable regions. However, in addition to the major PepM49 species, the pepsin digest of the type-49 streptococci also contained a smaller fragment, PepM49/a, as a minor component. Its formation was extremely sensitive to thepH of pepsin digestion. PepM49/a, which retains both the propensity to attain an alpha-helical conformation and the opsonic antibody epitope of the M49 molecule, contains only domains I and II like the other PepM proteins. Thus, as in the M5, M6, and M24 proteins, a pepsin cleavage site at the junction of the variable and conserved regions is indeed present in the M49 molecule, but is much less accessible relative to the other serotypes. Thus, the pepsin cleavage sites in the M protein correlate quite well with the boundaries of structurally distinct domains reflected by the predictive analysis. These sites apparently represent the flexible/hinge regions of the molecule. PepM49/a is the least repetitive and the shortest of the M protein pepsin fragments isolated so far. These results suggest that the flexibility of the interdomain regions in M protein may be dependent on the molecular size of their variable domains. The placement of a more accessible hinge within the conserved part of the M49 molecule, rather than at the junction of the variable and conserved domains, suggests that a critical molecular size may be essential for the efficient functioning of the M molecule.

Reversible Dihydroxypropylation of Amino Groups in Proteins: Application in Primary Structural Studies of Streptococcal M-Proteins

M-protein of group A streptococcus is an immunologically diverse antiphagocytic determinant of the bacteria1. In order to better understand the structure-function relationships of the M-proteins, we undertook the determination of their primary structure. The Pep M5 protein, a biologically active, pepsin-derived N-terminal half of the type 5 M protein, was selected for the initial study2. Pep M5 protein contains 6 arginines, 35 lysines, and 47 glutamates, with no methionines and tryptophans, thus limiting the choice for obtaining large peptides for sequence studies to cleavage at its arginyl peptide bonds2. Though arginine specific clostripain appeared to be satisfactory initially, detailed studies of the peptides generated by clostripain digestion of Pep M5 protein revealed that in addition to the major arginine cleavages, digestion occurred at some of the lysine residues. Furthermore, clostripain failed to cleave some of the arginyl bonds quantitatively3. Thus, a better choice for obtaining arginyl peptides appeared to be to take advantage of the high specificity of tryptic cleavage after chemically modifying the e-amino groups of the Pep M5 protein4. The relatively high lysine content of M-protein makes it essential that the reagent used for the modification be very selective to the amino functions and also be capable of derivatizing the lysine residues of the protein completely.

NH2-Terminal Sequence of the Clostripain and NBS Peptides of Streptococcal M5 Protein Purified in One Step by HPLC

M protein, the antiphagocytic surface molecule of the Group A streptococcus is a type specific, imiriuno logic ally diverse molecule. We have shown that the molecular characteristics of M proteins and the seven residue periodicity within their partial sequences are similar to those found in tropomyosin suggesting an alpha-helical coiled-coil structure for these molecules (1, 2). To determine if the seven residue periodicity extends throughout the molecule, sequence studies have been undertaken on a biologically active 19,000 dalton M protein fragment, namely Pep M5, isolated by limited proteolysis of the type 5 streptococci with pepsin (3).