alpha 1-Antitrypsin Christchurch, 363 Glu----Lys: mutation at the P'5 position does not affect inhibitory activity

alpha 1-Antitrypsin Christchurch was isolated from the plasma of a Cambodian woman who was heterozygous for this variant and for the normal M protein. Tryptic peptide maps revealed that the inhibitory-site peptide, 359-365 Ser-Ile-Pro-Pro-Glu,Val,Lys, was missing and replaced by two new peptides Ser-Ile-Pro-Pro,Lys and Val-Lys, indicating a mutation of 363 Glu----Lys. There was no obvious clinical condition associated with this new antitrypsin. Competition experiments showed that antitrypsin Christchurch reacted at the same rate as normal antitrypsin in the presence of limiting amounts of trypsin, chymotrypsin, thrombin and neutrophil elastase. Both inhibitors were inactivated by catalytic amounts of papain. This inactivation was due to cleavage at the phenylalanine residue at the P7 position, seven residues towards the N-terminal of the inhibitory site. A one-step ethanol extraction procedure is described for isolating the papain cleavage products.     

Identification of a second mutation in the protein-coding sequence of the Z type alpha 1-antitrypsin gene

This study reports the entire nucleotide sequence of the protein coding region sequence of the alpha 1-antitrypsin (alpha 1AT) Z gene, a common form of the alpha 1AT gene associated with serum alpha 1AT deficiency. In addition to Glu342 to Lys342 mutation in exon V which has been previously identified by peptide analysis, another point mutation (GTG to GCG in exon III) in the gene sequence predicts a second amino acid substitution (Val213 to Ala213) in the Z protein. This Val213 to Ala213 mutation was confirmed to be a general finding in Z type alpha 1AT gene by evaluating genomic DNA from 40 Z haplotypes using synthetic oligonucleotide gene probes directed toward the mutated exon III sequences in the Z gene. Furthermore, the exon III Val213 to Ala213 mutation eliminates a BstEII restriction endonuclease site in the alpha 1AT Z gene, allowing rapid identification of this Val213 to Ala213 substitution at the genomic DNA level. Surprisingly, when genomic DNA samples from individuals thought to be homozygous for the M1 gene (the most common alpha 1AT normal haplotype) were evaluated with BstEII, 23% of the M1 haplotypes were BstEII site negative, thus identifying a new form of M1 (i.e. M1(Ala213], likely identical to M1 but with an isoelectric focusing "silent" amino acid substitution (Val213 to Ala213). Although the relative importance of the newly identified exon III Val213 to Ala213 mutation to the pathogenesis of the abnormalities associated with the Z gene is not known, it is likely that M1(Ala213) gene represents a common "normal" polymorphism of the alpha 1AT gene that served as an evolutionary intermediate between the M1(Val213) and Z genes.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Mutation of asparagine 52 to glycine promotes the alkaline form of iso-1-cytochrome c and causes loss of cooperativity in acid unfolding

The kinetics and thermodynamics of the alkaline and acid conformational transitions of a Lys 79 --> Ala/Asn 52 --> Gly (A79G52) variant of iso-1-cytochrome c are studied. The Lys 79 --> Ala mutation is designed to limit heme ligation in the alkaline conformer to Lys 73. The Asn 52 --> Gly mutation is intended to shift the population of the alkaline conformer to physiological pH based on the hierarchical nature of the cooperative substructures of this protein. The midpoint pH for formation of the alkaline conformer is approximately 7.45. The kinetics for the alkaline conformational transition of the A79G52 variant are consistent with the ionization constant, pK(H), for the trigger group controlling formation of the alkaline conformer being approximately 9.5. This pK(H) is low for alkaline conformers involving lysine-heme ligation but is consistent with the pK(a) of the highest of three ionizable groups which modulate formation of the histidine-heme alkaline conformer of a His 73 variant of iso-1-cytochrome c [Martinez, R. E., and Bowler, B. E. (2004) J. Am. Chem. Soc. 126, 6751-6758]. The acid transition of the A79G52 variant is split into two phases. Both the Lys 79 --> Ala and Asn 52 --> Gly mutations are expected to affect the buried hydrogen bond network of cytochrome c, suggesting that this network is an important modulator of the acid unfolding of cytochrome c.     

Structure of Somatostatin Isolated from Bovine Retina

Abstract: Somatostatin-like immunoreactivity (SLI) from bovine retina was purified and its structure determined. Retinal tissue (1868 g) extracted with 3% acetic acid yielded 18.6 nmol SLI. This peptide was purified by chromatography on an affinity column made with anti-somatostatin antiserum, a reverse-phase C-18 HPLC column, and three sequential applications on a reverse-phase phenyl HPLC column. The peptide was purified 103,000-fold from the initial extract with an overall yield of 14.4%. Amino acid sequence determination by an automatic Edman degradation technique revealed the sequence to be as follows: Ser - Ala - Asn - Ser - Asn - Pro - Ala - Met - Ala - Pro - Arg - Glu - Arg - Lys - Ala - Gly - (Cys) - Lys - Asn - Phe - Phe - Trp - Lys - Thr - (Phe, Thr, Ser, Cys). The apparent identity of this peptide with somatostatin octacosapeptide (S28) purified from other mammalian tissue indicates the phylogenetic conservation of its structure and facilitates the use of the retina as a model system for studying the neurotransmitter function of somatostatin.     

Mutational investigation of the specificity determining region of the Src SH2 domain

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.     

Direct identification of lysine-33 as the principal cationic center of the omega-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator.

We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.     

The amino acid substitutions of human alpha 1-antitrypsin M3, X and Z

alpha 1-Antitrypsin has been isolated from individuals with inherited genetic variants M3, X and Z. A fragmentation and peptide mapping system is described which together with amino acid and sequence analyses revealed the substitutions in M3 at 376 of Glu to Asp, in X at 204 of Glu to Lys and in the physiologically innocent Z a mutation at 213 of Val to Ala. The latter represents a second amino acid substitution in the Z protein.     

Detection of a genetic variant, lysine→glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330–446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine→glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.     

The molecular defect of albumin Tagliacozzo: 313 Lys----Asn

Albumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299-329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys----Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Expression of a normal and variant Alzheimer's beta-protein gene in amyloid of hereditary cerebral hemorrhage, Dutch type: DNA and protein diagnostic assays

Amyloid fibrils deposited in cerebral vessel walls in Dutch patients with hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) are formed by polymerization of a 39-residue peptide similar to the beta-protein of Alzheimer's disease, Down syndrome, sporadic cerebral amyloid angiopathy and normal aging. Sequence analysis of genomic DNA in HCHWA-D patients demonstrated a point mutation, cytosine for guanine at position 1852 of the precursor beta-protein gene, which causes a single amino acid substitution (glutamine for glutamic acid) corresponding to position 22 of the amyloid protein. The normal allele was also present in these patients. To examine the expression of normal and variant beta-protein alleles in HCHWA-D we analyzed all the tryptic peptides obtained from several amyloid fractions from leptomeningeal vascular walls. Amino acid sequence of two peptides (T3a and T3b) with identical amino acid composition revealed that T3a had glutamine and T3b had glutamic acid at position 22. Thus both the normal and variant Alzheimer's beta-protein alleles are expressed in vascular amyloid in HCHWA-D and may be detected by tryptic peptide mapping. Moreover, we have developed a diagnostic assay for high risk populations and prenatal evaluation that is based on the existence of the mutation.     

An alpha 1-antitrypsin variant, Pi B Alhambra (Lys to Asp, Glu to Asp), with rapid anodal electrophoretic mobility.

A variant of human alpha 1-antitrypsin (alpha 1 AT) was found by acid starch gel electrophoresis and by thin-layer electrofocusing. The variant has an anodal migration velocity almost identical to PiB. It is designated as Pi B Alhambra. Pi B Alhambra was purified to homogeneity from a heterozygous PiM1/PiB Alhambra subject. Specific trypsin inhibitory activity and composition of amino acids and carbohydrates were similar to those of normal PiM1. The structural difference between the normal and the variant inhibitors was elucidated by peptide mapping of their tryptic digests. Two amino acid substitutions, Lys to Asp and Glu to Asp, were found. The amino acid substitution, Gly to Asp, has been found in a common PiM2 variant [1]. The Pi B Alhambra variant presumably originated by two steps of mutation: generation of PiM2 from wild type PiM1 by the substitution Gly to Asp, and subsequent generation of Pi B Alhambra from PiM2 by another substitution, Lys to Asp.     

Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal.

The minimal sequence requirement for a peroxisome-targeting signal was investigated using an in vitro import system. Carboxyl-terminal sequences Ser-Lys-Leu (SKL) and Leu-Gln-Ser-Lys-Leu (LQSKL) of acyl-CoA oxidase (AOX) directed to peroxisomes the fused proteins with import-incompetent forms of AOX and catalase that had been truncated, implying that the SKL tripeptide functions as a targeting signal. Elimination of the entire SKL sequence or deletion of any 1 or 2 amino acids in the sequence abolished the import activity of AOX. Substitution of alanine for serine did not affect the import activity. Topogenic activity was retained when lysine was mutated to either arginine or histidine, whereas mutation to glutamic acid completely abolished the activity. A synthetic peptide comprising the carboxyl-terminal 10 amino acid residues of AOX inhibited the import of the authentic AOX polypeptide, whereas other peptides in which SKL was mutated, deleted, or internally located were not effective. The uptake of AOX was little affected by the peptide with an amidated alpha-carboxyl group. These results strongly suggest that the carboxyl-terminal SKL motif sequence (Ser/Ala)-(Lys/Arg/His)-Leu functions as a topogenic signal in translocation of proteins into peroxisomes, requiring the whole tripeptide sequence with a free alpha-COOH group at the carboxyl terminus.     

A mutation in actin associated with neoplastic transformation

A new protein was recognized in a chemically transformed human fibroblast cell line when its proteins labeled with [35S]methionine were compared with those from normal human fibroblasts by two-dimensional gel electrophoresis. The new protein was found in the Triton-insoluble cytoskeletal fraction as well as in the Triton-soluble fraction, and it migrated very closely to beta- and gamma-actins on the gels. This new protein was identified as a variant form of actin by its reaction with antiactin antibody and its tryptic peptide pattern, which was identical to actin. mRNA coding for the variant actin was detected only in this particular transformed line. The size and cross- hybridizability with Dictyostelium actin cDNA of mRNA coding for the variant actin and complete amino acid sequence of the variant actin indicate that the new variant actin is the product of a mutated beta-actin gene. Only a single amino acid (glycine) at position 244 was replaced by aspartic acid. This substitution corresponds to a GC----AT transition, a point mutation. On the other hand, a highly malignant cell variant was isolated from the transformed line. The mutated beta-actin was further altered in this highly malignant subclone: it showed a more negative charge, rapid synthetic rate, and a short half-life in the cells. Incorporation into the cytoskeleton was significantly reduced in the mutated beta-actin. A hypothesis on the relationship between a mutation in the actin gene and oncogenic transformation was proposed.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.     

Multiple forms of histone H2B from the nematode Caenorhabditis elegans.

The complete amino acid sequence of histone H2B from the nematode Caenorhabditis elegans was determined. The protein as obtained by us is a mixture of multiple forms. Approx. 90% of the molecules consist of a polypeptide chain of 122 amino acids with alanine as N-terminal residue and proline at the second position. In the remaining 10% alanine is lacking and the chain starts with proline. In addition to the heterogeneity of chain length, polymorphism occurs at the positions 7 (Ala/Lys), 14 (Ala/Lys) and 72 (Ala/Ser) of the major chain and at position 6 (Ala/Lys) of the shorter chain. In the N-terminal third of the molecule there is a high degree of sequence homology to the corresponding region in H2B from Drosophila (insect), Patella (mollusc) and Asterias (starfish). In contrast, this part of the molecule differs considerably from mammalian histone H2B.     

Stability-increasing mutants of glucose dehydrogenase from Bacillus megaterium IWG3

A glucose dehydrogenase gene was isolated from Bacillus megaterium IWG3, and its nucleotide sequence was identified. The amino acid sequence of the enzyme deduced from the nucleotide sequence is very similar to the protein sequence of the enzyme from B. megaterium M1286 reported by Jany et al. (Jany, K.-D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) FEBS Lett. 165, 6-10). The isolated gene was mutagenized with hydrazine, formic acid, or sodium nitrite, and 12 clones (H35, H39, F18, F20, F191, F192, N1, N13, N14, N28, N71, and N72) containing mutant genes for thermostable glucose dehydrogenase were obtained. The nucleotide sequences of the 12 genes show that they include 8 kinds of mutants having the following amino acid substitutions: H35 and H39, Glu-96 to Gly; F18 and F191, Glu-96 to Ala; F20, Gln-252 to Leu; F192, Gln-252 to Leu and Ala-258 to Gly; N1, Glu-96 to Lys and Val-183 to Ile; N13 and N14, Glu-96 to Lys, Val-112 to Ala, Glu-133 to Lys, and Tyr-217 to His; N28, Glu-96 to Lys, Asp-108 to Asn, Pro-194 to Gln, and Glu-210 to Lys; and N71 and N72, Tyr-253 to Cys. These mutant enzymes have higher stability at 60 degrees C than the wild-type enzyme. The results of this study indicate that the tetrameric structure of glucose dehydrogenase is stabilized by several kinds of mutation, and at least one of the following amino acid substitutions stabilizes the enzyme: Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, and Tyr-253 to Cys.     

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function

We show that a synthetic peptide corresponding to the sequence of one putative Ca2+ binding motif of the cell adhesion molecule uvomorulin is able to complex Ca2+. This function is abolished if the first Asp in the peptide is replaced by Lys. Accordingly, we expressed in L cells mutant uvomorulin with a replacement of Asp to Lys or Ala. Mutant protein was resistant to Ca2+/trypsin under mild conditions but became susceptible at or near the site of replacement at higher concentrations, leaving the remaining Ca2+ binding domains protected. Remarkably, in cell aggregation assays both mutant uvomorulins failed to mediate cell adhesiveness, demonstrating that a single amino acid substitution in one Ca2+ binding site inactivates the adhesive function.