Complementary DNA sequence of lamprey fibrinogen beta chain

The cDNA sequence of the beta chain of lamprey fibrinogen has been determined. To that end, an oligonucleotide probe was synthesized that corresponded to an amino acid sequence from the carboxy-terminal region of the lamprey fibrinogen beta chain. The insert actually began with residue 3 of the fibrin beta chain; it ran through to a terminator codon following the carboxy-terminal residue at position 443 and then continued for an additional 606 nucleotides of noncoding sequence to its 3' end. The inferred amino acid sequence was verified by comparison with assorted cyanogen bromide fragments isolated from the beta-chain protein, including two carbohydrate-containing peptides that corresponded to segments containing the carbohydrate-attachment consensus sequence. Overall, the lamprey chain is 49% identical with the beta chain from human fibrinogen. This is the same degree of resemblance as was found for the lamprey and human gamma chains. Moreover, the principal regions of conservation are the same in both the beta and gamma chains. Differences and similarities in the physiological behavior of the two fibrinogens are assessed in terms of the observed amino acid replacements.     

Interaction of fibrin(ogen) with heparin: further characterization and localization of the heparin-binding site

The beta chain 15-42 sequence of the fibrin(ogen) E region was implicated in heparin binding [Odrljin et al. (1996) Blood 88, 2050-2061]; whether heparin binds to other fibrin(ogen) regions remains to be clarified. To address this question, we studied the interaction of heparin with fibrinogen, fibrin, and their major fragments D(1), D-D, E(1), E(3), and alphaC, which together cover the entire structure of the molecule, by ligand blotting, surface plasmon resonance, and fluorescence. All three techniques revealed that at physiological ionic conditions only fibrin(ogen) and the E(1) fragment bind heparin, indicating that the only physiologically relevant heparin-binding site of fibrin(ogen) is located in its E region. To test whether the beta15-42 sequence is sufficient to form this site or some additional sequences are also involved, we tested the interaction of heparin with a number of beta15-42-containing fragments. The synthetic beta15-42 peptide bound heparin weakly (K(d) = 44.5 microM) while the recombinant beta15-57 and beta15-64 fragments exhibited almost 7-fold higher affinity (K(d) = 6.4 and 7.1 microM, respectively), indicating that the beta43-57 region is also important for heparin binding. At the same time the recombinant dimeric disulfide-linked (beta15-66)(2) fragment which mimics the dimeric arrangement of the beta chains in fibrin bound heparin with high affinity (K(d) = 66 nM), almost 100-fold higher than that for the monomeric fragments. This affinity was similar to those determined for fibrin and the E(1) fragment (K(d) = 72 and 70 nM, respectively) suggesting that (beta15-66)(2) mimics well the heparin-binding properties of the latter two. Altogether, these results indicate that the only heparin-binding site in fibrin(ogen) is formed by NH(2)-terminal portions of the beta chains, including residues beta15-57, and that dimerization is essential for high-affinity binding.     

Identification of B beta chain domains involved in human fibrinogen assembly.

Fibrinogen chains are assembled in a stepwise manner in the rough endoplasmic reticulum prior to secretion of the final six-chain dimeric molecule. Previous studies indicated that the synthesis of B beta may be a rate-limiting factor in the assembly of human fibrinogen. To determine the domains of B beta which interact with the other two component chains of fibrinogen, deletion mutants of B beta were transiently co-expressed, together with A alpha and gamma chains, in COS cells, and fibrinogen assembly and secretion were measured. Deletion of the COOH-terminal half of the B beta chain (amino acids 208-461) did not affect assembly and secretion. Assembly of A alpha, gamma, and B beta also occurred when the first NH2-terminal 72 amino acids of B beta were deleted, but not when 93 amino acids were deleted. This indicates that the B beta domain between amino acids 73 and 93 is necessary for the assembly of the three fibrinogen chains. This domain marks the start of the alpha-helical "coiled-coil" region of fibrinogen.     

Complete sequence of the lamprey fibrinogen alpha chain

The complete amino acid sequence of the lamprey fibrinogen alpha chain has been determined by a combination of peptide sequencing and cDNA and genomic cloning. The chain, which has an apparent molecular weight by dodecyl sulfate-polyacrylamide gel electrophoresis of ca. 100,000, is composed of 961 amino acid residues and has a calculated molecular weight of 96,722. It is distinguished by a large number of 18-residue repeats in a region where mammalian fibrinogens have 13-residue repeats. The data are in accord with our previous finding that the lamprey alpha chain has a distinctive amino acid composition, almost half the residues being glycine, serine, or threonine. The chain differs from mammalian alpha chains in that there are no cysteines in the carboxy-terminal half, and thus no intrachain loop, nor are there any RGD sequences in the lamprey alpha chain. Taken together with previous data on the sequences of the beta and gamma chains, the findings bear significantly on our understanding of fibrin formation. The alpha chain also provides an interesting case of structural convergence during evolution.     

Structural basis for the species selectivity of a fibrin-specific monoclonal antibody

The structural determinant underlying the species specificity of a monoclonal anti-fibrin antibody (59D8) is the leucyl residue at position 5 in beta-chains of human fibrin. Anti-fibrin antibody 59D8 which had been elicited by immunization with human beta(1-7) peptide, Gly-His-Arg-Pro-Leu-Asp-Lys, binds to human and canine fibrins but not to bovine, ovine, or porcine fibrins. A comparison of the available amino acid sequence data suggested that the ability of anti-fibrin antibody 59D8 to discriminate among various fibrin beta-chains might be due to the amino acid at position 5. This was confirmed by competitive inhibition studies using synthetic fibrin-like peptides and determination of the amino acid sequences of the N-termini of ovine and porcine fibrin beta-chains. Edman degradation employing o-phthalaldehyde blocking permitted use of fibrin monomer rather than its separated constituent polypeptide chains. The same sequencing strategy was used to obtain partial sequence data for the alpha-chains of bovine, ovine, and porcine fibrin.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

A cluster of basic amino acid residues in the gamma370-381 sequence of fibrinogen comprises a binding site for platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa)

Adhesive interactions of platelet integrin alpha(IIb)beta3 with fibrinogen and fibrin are central events in hemostasis and thrombosis. However, the mechanisms by which alpha(IIb)beta3 binds these ligands remain incompletely understood. We have recently demonstrated that alpha(IIb)beta3 binds the gamma365-383 sequence in the gammaC-domain of fibrin(ogen). This sequence contains neither the AGDV nor the RGD recognition motifs, known to bind alpha(IIb)beta3, suggesting the different specificity of the integrin. Here, using peptide arrays, mutant fibrinogens, and recombinant mutant gammaC-domains, we have examined the mechanism whereby alpha(IIb)beta3 binds gamma365-383. The alpha(IIb)beta3-binding activity was localized within gamma370-381, with two short sequences, gamma370ATWKTR375 and gamma376WYSMKK381, being able to independently bind the integrin. Furthermore, recognition of alpha(IIb)beta3 by gamma370-381 depended on four basic residues, Lys373, Arg375, Lys380, and Lys381. Simultaneous replacement of these amino acids and deletion of the gamma408AGDV411 sequence in the recombinant gammaC-domain resulted in the loss of alpha(IIb)beta3-mediated platelet adhesion. Confirming the critical roles of the identified residues, abnormal fibrinogen Kaiserslautern, in which gammaLys380 is replaced by Asn, demonstrated delayed clot retraction and impaired alpha(IIb)beta3 binding. Also, a mutant recombinant fibrinogen modeled after the naturally occurring variant Osaka V (gammaArg375 --> Gly) showed delayed clot retraction and reduced binding to purified alpha(IIb)beta3. These results identify the gamma370-381 sequence of fibrin(ogen) as the binding site for alpha(IIb)beta3 involved in platelet adhesion and clot retraction and define the new recognition specificity of this integrin.     

Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta - and gamma -chains

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.     

Beta(Leu121-Lys122) segment of fibrinogen is in a region essential for plasminogen binding by fibrin fragment E

It was shown previously that two sequentially nonidentical regions of human fibrin(ogen), present in fragments D and E, carry specific plasminogen-binding sites [V aradi , A., & Patthy , L. (1983) Biochemistry 22, 2440-2446]. Comparison of the affinity of a variety of fragment E species for immobilized Lys-plasminogen revealed that fragment E3e [(alpha 20/24-78, beta 54-122, gamma 1-53)2] possesses a strong plasminogen-binding site, whereas fragment E3t [(alpha 20/24-78, beta 54-120, gamma 1-53)2] has 30-fold lower affinity for the affinant . Since the two fragments differ only in the beta ( Leu121 - Lys122 ) segment, this suggests that residues beta ( Leu121 - Lys122 ), present in the triple-helical connector region of fibrin(ogen), are essential for plasminogen binding by fragment E. Reduction and alkylation of fragment E3e lead to the destruction of the plasminogen-binding site, indicating that none of the separated, alkylated polypeptide chains of the fragment are able to bind to plasminogen and probably the coiled-coil superstructure of the connector region is necessary for the maintenance of the plasminogen-binding site of fragment E.     

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.     

A pentapeptide from the laminin B1 chain mediates cell adhesion and binds the 67,000 laminin receptor

Laminin promotes epithelial cell adhesion in part through a site of nine amino acids CDPGYIGSR on the B1 chain. Using smaller synthetic peptides from this sequence as well as various peptides with amino acid substitutions, we find that the minimum sequence necessary for efficient cell adhesion as well as receptor binding is YIGSR. The deletion of tyrosine or the substitution of arginine in the peptides resulted in a significant loss of activity. The presence of an amide group on the terminal arginine of either peptide increases activity significantly. YIGSR is active in promoting the adhesion of a variety of epithelial cells; however, it is inactive with chondrocytes, fibroblasts, and osteoblasts.     

Lamprey fibrinopeptide B is A glycopeptide

One of the peptides released from lamprey fibrinogen during its transformation into fibrin has been found to contain covalently bound carbohydrate. The peptide, which also contains tyrosine O-sulfate, corresponds to the mammalian fibrinopeptide B and is the amino-terminal segment of the lamprey fibrinogen β-chain. As noted previously, this peptide is the only one released when lamprey fibrinogen is coltted by mammalian thrombin. Of the more than fifty sets of fibrinopeptides characterized from various species, this is the first one found to contain carbohydrate.     

Tissue-type plasminogen activator is a multiligand cross-beta structure receptor

Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA, but the structural basis for this interaction is unknown. Amyloid beta (Abeta) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimer's disease. Like fibrin, it stimulates tPA-mediated plasmin formation. Intermolecular stacking of peptide backbones in beta sheet conformation underlies cross-beta structure in amyloid peptides. We show here that fibrin-derived peptides adopt cross-beta structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Abeta and islet amyloid polypeptide (IAPP) (associated with pancreatic beta cell toxicity in type II diabetes), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-beta structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-beta structure is the common denominator in tPA binding ligands.     

Fibrin(ogen)-alpha M beta 2 interactions regulate leukocyte function and innate immunity in vivo

In addition to its well-characterized role in hemostasis, fibrin(ogen) has been proposed to be a central regulator of the inflammatory response. Multiple in vitro studies have demonstrated that this hemostatic factor can alter leukocyte function, including cell adhesion, migration, cytokine and chemokine expression, degranulation, and other specialized processes. One important link between fibrin(ogen) and leukocyte biology appears to be the integrin receptor alpha(M)beta(2)/Mac-1, which binds to immobilized fibrin(ogen) and regulates leukocyte activities. Although it is well established that fibrin(ogen) is a ligand for alpha(M)beta(2), the precise molecular determinants that govern this interaction are only now becoming clear. A novel line of mice expressing a mutant form of fibrinogen (Fib gamma(390-396A)) has revealed that gamma chain residues 390-396 are important for the high-affinity engagement of fibrinogen by alpha(M)beta(2) and leukocyte function in vivo. Fibrinogen gamma(390-396A) failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, monocytes, and macrophages, and mice expressing this fibrinogen variant were found to exhibit a major defect in the host inflammatory response following acute challenges. Most notably, Fib gamma(390-396A) mice display a profound impediment in Staphylococcus aureus elimination by leukocytes following intraperitoneal inoculation. These findings have positively established the physiological importance of fibrin(ogen) as a ligand for alpha(M)beta(2) and illustrate that the fibrin(ogen) gamma chain residues 390-396 constitute a critical feature of the alpha(M)beta(2) binding motif. Finally, the Fib gamma(390-396A) mice represent a valuable system for better defining the contribution of fibrin(ogen) to the inflammatory response in the absence of any confounding alteration in clotting function.     

Probing interactions between the coagulants thrombin, Factor XIII, and fibrin(ogen)

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.     

Delineation and conformational analysis of two synthetic peptide models of antigenic sites on rodent cytochrome c

Two regions of rodent cytochrome c, one within the first four residues of the molecule, which is N-acetylated, and one at a beta bend around residue 44, are known to be immunogenic and antigenic in rabbits. Using sequential peptide synthesis, we have determined the residues required for linear synthetic peptides within these sequences to bind to antibody raised in rabbits to intact rat cytochrome c. The residues that were important in binding the N-terminal peptides were N-acetylglycine at position 1 and valine at position 3. The smallest peptide sequence around residue 44 that would bind to antibodies was Gln-Ala-Ala-Gly-Phe. A theoretical conformational analysis of these peptides showed that the amino-terminal tetrapeptide adopts a wide statistical ensemble of conformational states and that the addition of residues beyond 41 and 45 in the other sequence does not appear to stabilize longer peptides in the native beta-bend conformation. Thus, the antigenicity conferred by Phe-46 and Gln-42 in this peptide is most likely due to the direct interaction of the side chains of these residues with the antibody binding site. The demonstration here that native conformation is not essential for antigenic peptides to bind to antibodies raised against the whole protein indicates that the association energy between antigen and antibody can be sufficient to induce conformation in conformationally flexible peptides. This supports the concept that anti-protein and anti-peptide antibodies may invoke conformational changes in cross-reactive protein antigens and may explain why longer peptides, which may adopt stable nonnative secondary structure, often do not bind to antibodies raised to the whole molecule.     

Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation

We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha(IIb)beta(3), based on absence of retraction of alpha(IIb)beta(3)-deficient thrombasthenic platelets. However, only a subset of alpha(IIb)beta(3)-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha(IIb)beta(3) in fibrin clot retraction versus aggregation. Immunoblotting demonstrated a phosphorylated protein pattern comparable with aggregation at early time points. However, as opposed to aggregation, tyrosine phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was alpha(IIb)beta(3)-dependent, since it was blocked by alpha(IIb)beta(3)-specific inhibitors and was absent in thrombasthenic platelets. Inhibition of platelet clot retraction by phenyl-arsine oxide and peroxovanadate, suggested a role for tyrosine phosphatases. Cytochalasin D and E (5 microm) blocked fibrin clot retraction and tyrosine dephosphorylation, suggesting regulation by actin cytoskeleton assembly. Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were alpha(IIb)beta(3)-dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that alpha(IIb)beta(3) engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.     

Amino-aromatic interactions in proteins

Geometric analysis of 33 refined high-resolution protein crystal structures (2 A or higher) demonstrates that side-chain amino groups interact with aromatic side chains. Positively charged or delta(+) amino groups of lysine, arginine, asparagine, glutamine and histidine are preferentially located within 6 A of the ring centroids of phenylalanine, tyrosine and tryptophan, where they make van der Waals' contact with the delta(-) pi-electrons and avoid the delta(+) ring edge. This geometric pattern is different from the distribution expected due to random close packing of side chains in a protein. It is opposite to oxygen- and sulfur-aromatic interactions, similar to aromatic-aromatic interactions, and almost certainly electrostatic in origin.     

Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface-associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal alpha-enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA). The C-terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)-binding site of Eno by investigating the protein-protein interaction. Plasmin(ogen)-binding activity of C-terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno-plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248-256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)-binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing alpha-enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen-binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen-binding motif in the pathogenesis of pneumococcal diseases.     

Amino acid specificity of glycation and protein-AGE crosslinking reactivities determined with a dipeptide SPOT library.

Advanced glycation end products (AGEs) contribute to changes in protein conformation, loss of function, and irreversible crosslinking. Using a library of dipeptides on cellulose membranes (SPOT library), we have developed an approach to systematically assay the relative reactivities of amino acid side chains and the N-terminal amino group to sugars and protein-AGEs. The sugars react preferentially with cysteine or tryptophan when both the alpha-amino group and the side chains are free. In peptides with blocked N-terminus and free side chains, cysteine, lysine, and histidine were preferred. Crosslinking of protein-AGEs to dipeptides with free side chains and blocked N termini occurred preferentially to arginine and tryptophan. Dipeptide SPOT libraries are excellent tools for comparing individual reactivities of amino acids for nonenzymatic modifications, and could be extended to other chemically reactive molecules.