Genomic profiling in Down syndrome acute lymphoblastic leukemia identifies histone gene deletions associated with altered methylation profiles.

Patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have distinct clinical and biological features. Whereas most DS-ALL cases lack the sentinel cytogenetic lesions that guide risk assignment in childhood ALL, JAK2 mutations and CRLF2 overexpression are highly enriched. To further characterize the unique biology of DS-ALL, we performed genome-wide profiling of 58 DS-ALL and 68 non-DS (NDS) ALL cases by DNA copy number, loss of heterozygosity, gene expression and methylation analyses. We report a novel deletion within the 6p22 histone gene cluster as significantly more frequent in DS-ALL, occurring in 11 DS (22%) and only 2 NDS cases (3.1%) (Fisher's exact P=0.002). Homozygous deletions yielded significantly lower histone expression levels, and were associated with higher methylation levels, distinct spatial localization of methylated promoters and enrichment of highly methylated genes for specific pathways and transcription factor-binding motifs. Gene expression profiling demonstrated heterogeneity of DS-ALL cases overall, with supervised analysis defining a 45-transcript signature associated with CRLF2 overexpression. Further characterization of pathways associated with histone deletions may identify opportunities for novel targeted interventions.

A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen.

Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.

SdrG, a fibrinogen-binding bacterial adhesin of the microbial surface components recognizing adhesive matrix molecules subfamily from Staphylococcus epidermidis, targets the thrombin cleavage site in the Bbeta chain.

Staphylococcus epidermidis is an important opportunistic pathogen and is a major cause of foreign body infections. We have characterized the ligand binding activity of SdrG, a fibrinogen-binding microbial surface component recognizing adhesive matrix molecules from S. epidermidis. Western ligand blot analysis showed that a recombinant form of the N-terminal A region of SdrG bound to the native Bbeta chain of fibrinogen (Fg) and to a recombinant form of the Bbeta chain expressed in Escherichia coli. By analyzing recombinant truncates and synthetic peptide mimetics of the Fg Bbeta chain, the binding site for SdrG was localized to residues 6-20 of this polypeptide. Recombinant SdrG bound to a synthetic 25-amino acid peptide (beta1-25) representing the N terminus of the Fg Bbeta chain with a KD of 1.4 x 10(-7) m as determined by fluorescence polarization experiments. This was similar to the apparent K(D) (0.9 x 10(-7) m) calculated from an enzyme-linked immunosorbent assay where SdrG bound immobilized Fg in a concentration-dependent manner. SdrG could recognize fibrinopeptide B (residues 1-14), but with a substantially lower affinity than that observed for SdrG binding to synthetic peptides beta1-25 and beta6-20. However, SdrG does not bind to thrombin-digested Fg. Thus, SdrG appears to target the thrombin cleavage site in the Fg Bbeta chain. In fact, SdrG was found to inhibit thrombin-induced fibrinogen clotting by interfering with fibrinopeptide B release.

Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1.

Integrins alpha(1)beta(1) and alpha(2)beta(1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha(1)beta(1) (alpha(1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999) J. Biol. Chem. 274, 24906-24913). Here, we compared the binding of the recombinant A-domain of alpha(2)beta(1) (alpha(2)A) to type I collagen with that of alpha(1)A using surface plasmon resonance and showed that alpha(2)A exhibited only one detectable class of binding sites in type I collagen, with a K(D) of approximately 10 microm at approximately 3 binding sites per collagen molecule. We further demonstrated that alpha(1)A and alpha(2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha(1)A- and alpha(2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha(1)A and alpha(2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha(1)A and alpha(2)A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of alpha(1)A and alpha(2)A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha(1)A and alpha(2)A.

The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen.

Staphylococcus aureus is an important pathogen capable of causing a wide spectrum of diseases in humans and animals. This bacterium expresses a variety of virulence factors that participate in the process of infection. These include MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that mediate the adherence of the bacteria to host extracellular matrix components, such as collagen, fibronectin (Fn), and fibrinogen (Fg). Two Fn-binding MSCRAMMs, FnbpA and FnbpB, have been previously identified. The Fn binding activity has been localized to the approximately 40-amino acid residue D repeats in the C-terminal part of these proteins. However, no biological activity has yet been attributed to the N-terminal A regions of these proteins. These regions exhibit substantial amino acid sequence identity to the A regions of other staphylococcal MSCRAMMs, including ClfA, ClfB, and SdrG (Fbe), all of which bind Fg. This raises the question of whether the Fn-binding MSCRAMMs can also bind specifically to Fg. In this report, we show that a recombinant form of the A region of FnbpA does specifically recognize Fg. We localize the binding site in Fg for recombinant FnbpA to the gamma-chain, in particular to the C-terminal residues of this polypeptide, the site also recognized by ClfA. In addition, we demonstrate that recombinant FnbpA can compete with ClfA for binding to both immobilized and soluble Fg. By the use of surface plasmon resonance spectroscopy and fluorescence polarization, we determine the dissociation equilibrium constant for the interaction of recombinant FnbpA with intact immobilized Fg and with a synthetic C-terminal gamma-chain peptide, respectively. Finally, by overexpressing FnbpA in a mutant strain of S. aureus that lacks the expression of both ClfA and ClfB, we show that native FnbpA can mediate the interaction of S. aureus with soluble Fg.

The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site.

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin alphaIIbbeta3 recognize the same domain at the extreme C terminus of the fibrinogen gamma-chain. alphaIIbbeta3 interaction with this domain is known to occur in close proximity to a Ca2+-binding EF-hand structure in the alpha-subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca2+-binding EF-hand-like motif at residues 310-321 within the fibrinogen-binding domain. Deletion mutagenesis and site-directed mutagenesis of this EF-hand in recombinant truncated ClfA proteins (Clf40, residues 40-559; and Clf41, residues 221-559) resulted in a significant reduction of affinity for native fibrinogen and a fibrinogen gamma-chain peptide. Furthermore, Ca2+ (or Mn2+) could inhibit the binding of the fibrinogen gamma-chain peptide to Clf40-(40-559) and the adhesion of S. aureus cells to immobilized fibrinogen with an IC50 of 2-3 mM. In contrast, Mg2+ (or Na+) at similar concentrations had no effect on the ClfA-fibrinogen interaction. Far-UV CD analysis of Clf40-(40-559) and Clf41-(221-559) in the presence of metal ions indicated Ca2+- and Mn2+-induced differences in secondary structure. These data suggest that Ca2+ binds to an inhibitory site(s) within ClfA and induces a conformational change that is incompatible with binding to the C terminus of the gamma-chain of fibrinogen. Mutagenesis studies indicate that the Ca2+-dependent inhibitory site is located within the EF-hand motif at residues 310-321.

Multiple specificities of the staphylococcal and streptococcal fibronectin-binding microbial surface components recognizing adhesive matrix molecules.

Many pathogenic gram-positive bacteria express fibronectin (Fn)-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), most of which have a similar structural organization with a primary ligand-binding domain consisting of 3-6 repeats of 40-50 amino-acid-residue motifs. The MSCRAMMs appear to preferentially bind to the N-terminal region of Fn, which is composed of five type-I modules. Here we report that the Fn-binding MSCRAMM FnbpA of Staphylococcus aureus contains a second ligand-binding domain located outside the repeat units. In addition, several sites in the Fn N-terminus presented as recombinant type-I module pairs bind to the repeat domain of the MSCRAMM. All of the MSCRAMMs analyzed, which include FnbpA of Staphylococcus aureus, Sfb of Streptococcus pyogenes, and FnbA and FnbB of Streptococcus dysgalactiae, were shown to bind to multiple sites in the N-terminal domain of Fn. By dissecting the repeat domain of FnbpA using synthetic peptides and recombinant fragments, we show that discrete, different motifs are responsible for the binding to individual sites in Fn, rather than a common motif being able to bind to several pairs of type-I Fn modules. The C-terminal half of many of the MSCRAMM repeat units contain a common motif, which is shown here to bind to the type-I module pair 4 and 5. In addition, some of the repeat units of FnbpA contain N-terminal motifs which bound to the type-I module pairs 1-2 and 2-3, respectively. These latter binding motifs appear to be partly overlapping and dependent on flanking sequences. Fluorescence polarization experiments using fluorescein-labeled MSCRAMM peptides and recombinant type-I Fn module pairs revealed dissociation constants of 1-13 microM. It was also shown that the fluorescein-labeled peptides differed in their primary binding sites on Fn.

Critical residues in the ligand-binding site of the Staphylococcus aureus collagen-binding adhesin (MSCRAMM).

We have identified a discrete collagen-binding site within the Staphylococcus aureus collagen adhesin that is located in a region between amino acids Asp209 and Tyr233. Polyclonal antibodies raised against a recombinant form of the collagen adhesin inhibited the binding of collagen type II to S. aureus. When overlapping synthetic peptides mimicking segments of the adhesin fragment were tested for their ability to neutralize the inhibitory activity of the antibody only one peptide, CBD4 was found to be active. CBD4 bound directly to collagen and at high concentrations inhibited the binding of collagen to S. aureus. A synthetic peptide derivative of CBD4 lacking 2 carboxyl-terminal residues (Asn232, Tyr233) had no inhibitory activity. The importance of these residues for collagen binding was confirmed by biospecific interaction analysis. Mutant adhesin proteins N232-->A and Y233-->A exhibited dramatic changes in collagen binding activity. The dominant dissociation rate for the binding of mutant adhesin protein N232-->A to immobilized collagen II decreased almost 10-fold, while the Y233-->A and the double mutant exhibited even more significant decreases in affinity and apparent binding ratio when compared to the wild type protein.

Fibronectin receptors from gram-positive bacteria: comparison of active sites.

Many parasitic bacteria express fibronectin binding proteins that are located on the cell surface. These proteins may act as adhesins and mediate the adherence of the microorganisms to fibronectin-containing host tissues. The ligand binding sites in the fibronectin receptor proteins from Gram-positive bacteria are composed of unique 37-48 amino acid long motifs that are repeated 3-4 times. We have now expressed the ligand binding sites of fibronectin receptors from Staphylococcus aureus, Streptococcus dysgalactiae (two receptors), and Streptococcus pyogenes as recombinant proteins. The purified recombinant proteins have the expected molecular weights as indicated by electrospray mass spectroscopy although they migrate abnormally on SDS-PAGE. Each recombinant protein effectively inhibited the binding of 125I-labeled intact fibronectin or the N-terminal fibronectin domain to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus pyogenes. The relative inhibitory potency of the different recombinant proteins was similar for all target bacteria and is reflected in their relative affinities for fibronectin. Synthetic peptides corresponding to the repeat units of the ligand binding site of the fibronectin receptor proteins were shown to inhibit the binding of the N-terminal fibronectin fragment to Streptococcus pyogenes cells. Together with amino acid sequence comparison, these data demonstrate that the repeat motif of the fibronectin receptor of Streptococcus pyogenes conforms to the consensus sequence previously reported for the Staphylococcus aureus receptor and to one of the Streptococcus dysgalactiae receptors (McGavin et al., 1993).

Monoclonal antibody to the aminotelopeptide of type II collagen: loss of the epitope after stromelysin digestion.

A monoclonal antibody was prepared to the aminotelopeptide of type II collagen after immunization of DBA/1 mice with lathyritic type II collagen and subsequent screening for antibodies that recognize lathyritic but not pepsin-digested type II collagen. One antibody (called 5B2) was identified that recognized a short peptide sequence in the aminotelopeptide of chicken type II collagen but did not recognize other collagen types. Further characterization of the epitope was achieved using a Multipin system and the epitope was localized to a short linear sequence of six amino acids. The antibody recognized type II collagen from a variety of species including man and mouse. The epitope for 5B2 was found to be susceptible to cleavage with recombinant stromelysin without cleavage of the major collagen triple helix. Comparison was made between MAb 5B2 and two other antibodies (called MAb 2B1 and MAb 6B3) that recognize separate epitopes located along the triple helix of the type II collagen molecule.

Heparin-binding peptides from thrombospondins 1 and 2 contain focal adhesion-labilizing activity.

The cell adhesion regulating extracellular matrix glycoprotein, thrombospondin (TSP), causes a loss of focal adhesion plaques from spread endothelial cells and fibroblasts. To localize the site on TSP that has focal adhesion-labilizing activity, we initially tested proteolytic fragments of TSP for activity. The heparin-binding fragment has significant focal adhesion-labilizing activity, whereas the nonheparin-binding 140-kDa fragment had no significant activity. These results were consistent with previous data that showed that both a monoclonal antibody to the heparin-binding domain of TSP (A2.5) and heparin neutralized TSP activity. Peptides from putative heparin binding sequences of the amino-terminal heparin-binding domain of TSP were synthesized and tested for their ability to cause loss of focal adhesions. The hep I peptide (amino acids 17-35) caused maximal loss of focal adhesions and was active at 0.1 microM, whereas peptide hep II (74-95) and peptide hep III (170-189) were inactive. The activity of the hep I peptide was neutralized by the addition of heparin and heparan sulfate but not by chondroitin sulfate. The basic amino acids in the hep I sequence appear to be required for focal adhesion-labilizing activity, because modification of the lysine residues at amino acids 24 and 32 rendered the peptide completely inactive. In addition, a peptide from the analogous sequence of mouse TSP 2, in which basic residues are conserved, was nearly as active as hep I from TSP1. These data show that the anti-adhesive activity of TSP is conserved in both TSP1 and TSP2 and that the active site is located in a 19-amino acid sequence in the heparin-binding domain of TSPs.

Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus. Involvement of conserved residues in ligand binding.

The nucleotide sequence of two genes encoding fibronectin (Fn) receptors FnBA and FnBB of Streptococcus dysgalactiae S2 revealed the presence of repeated motifs (called RA1-A3 and RB1-B3, respectively) which encode Fn binding activity (Lindgren, P.-E., McGavin, M. J., Signäs, C., Guss, B., Gurusiddappa, S., Höök, M., and Lindberg, M. (1993) Eur. J. Biochem. 214, 819-827). Synthetic peptides of 32-37 amino acids, corresponding to individual repeated motifs, were assayed for the ability to inhibit Fn binding to cells of S. dysgalactiae. Within the RA motifs, peptide A2 was 10-fold more active than either A1 or A3, while in the RB motifs, only B3 was active. The same level of activity is observed when these synthetic peptides were assayed for inhibition of Fn binding to cells of Staphylococcus aureus. Likewise, synthetic peptides corresponding to the RD1-D3 motifs, which comprise a ligand binding domain in a Fn receptor from S. aureus, inhibit binding of Fn to both S. aureus and S. dysgalactiae. Assays of chemically modified peptides and peptide fragments derived from chemical or proteolytic cleavage suggest that a conserved core sequence, defined as ED(T/S) (X9,10)GG(X3,4)(I/V)DF, within a 30-amino acid-long segment is present in the active RA and RD motifs. Analyses of the importance of individual residues of this core sequence indicate that the ED(T/S) motif is nonessential, whereas the GG and the (I/V)DF together with additional acidic residues in the C-terminal half of the peptide are required for activity.

Two different genes coding for fibronectin-binding proteins from Streptococcus dysgalactiae. The complete nucleotide sequences and characterization of the binding domains.

The binding of Streptococcus dysgalactiae to fibronectin involves fibronectin-binding protein(s) present on the bacterial surface. Previously, we reported the cloning of two different genes coding for cell-wall-associated fibronectin-binding proteins from S. dysgalactiae strain S2 [Lindgren, P.-E., Speziale, P., McGavin, M. J., Monstein, H.-J., Höök, M., Visai, L., Kostiainen, T., Bozzini, S. & Lindberg, M. (1992) J. Biol. Chem. 267, 1924-1931]. The two genes, fnbA and fnbB, have now been sequenced and the primary amino acid sequences of the two fibronectin-binding proteins, FnBA and FnBB, have been deduced. The two proteins have predicted molecular masses of 117 kDa and 122 kDa, respectively, and are organized in a similar way. The fibronectin-binding activities are localized in repeated motifs, 32-37 amino acids long, in the COOH-terminal regions of the proteins. The two fibronectin-binding proteins have heterologous amino acid sequences, except for the COOH-terminal ends which include the fibronectin-binding repeats. The fibronectin-binding regions of the genes have been fused to IgG-binding domains of protein A, utilizing the IgG-binding capacity of the resulting fusion proteins, to facilitate isolation of the fibronectin-binding domains.

Fibronectin binding determinants of the Staphylococcus aureus fibronectin receptor.

Synthetic peptide analogs mimicking a repeated motif within the Staphylococcus aureus fibronectin receptor inhibit binding of the bacteria to fibronectin (Signäs, C., Raucci, G., Jonsson, K., Lindgren, P. E., Anantharamaiah, G. M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). In this study, we have further localized the fibronectin-binding determinant within the 37 amino acid D3 peptide. Chemical modification of the carboxyl side chains of the glutamic and aspartic residues in D3 abolished fibronectin-binding activity, whereas modifications of lysine or tyrosine residues had little effect. An active peptide encompassing residues 15-36 was isolated from a trypsin digest of D3, and a synthetic peptide S16-36 had activity comparable with that of intact D3. Scrambling the amino acid sequence of S16-36 or replacing the aspartic and glutamic residues with asparagine and glutamine resulted in loss of activity. Therefore, one or more of the acidic residues are essential for activity. However, additional sequence is required. Reduction in the size of S16-36 from either the N- or C-terminal end resulted in peptides with greatly diminished activity. These data suggest that the amino acids essential for binding fibronectin are contained within residues 21-33 of the D3 peptide and that the flanking N- and C-terminal amino acids are necessary for the peptide to acquire a conformation that is favorable for fibronectin binding.

Synthesis and biological studies of dermorphin and its analogs substituted at positions 5 and 7.

Dermorphin and seven of its analogs substituted at positions 5 and/or 7, have been synthesized by the solid phase method employing mainly 9-fluorenylmethyloxycarbonylamino acid trichlorophenyl esters in presence of l-hydroxybenzotriazole, the solid support being the Merrifield resin. Among the analogs synthesized, the most interesting is [Tyr7]dermorphin. It is one of the most potent dermorphin analogs reported so far. Compared to the natural peptide, it is about two times more potent in the GPI (in vitro) and nearly 1.4 times more potent in its analgesic activity in mice by the hot plate test (in vivo). Further, its antidiarrhoeal activity in mice (in vivo) is comparable to that of dermorphin. On the other hand, [Thr7]dermorphin is almost as potent as dermorphin.