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PubMed2003

Design and evaluation of novel nucleoside analogs (WNA) for specific formation of non-natural type triplexes containing a TA or CG interrupting site.

In a search of suitable recognition molecules for the formation of stable non-natural triplex DNA, we have developed the new nucleoside analogs (WNA, W-shape nucleoside analog) bearing an aromatic ring, a recognition base and a bicyclic skeleton to hold them. A successful result with WNA-betaT that recognizes a TA base pair selectively has suggested that the basic structure of the WNA is useful as a scaffold for search of other candidates for the formation of triplex containing a TA or a CG interrupting site. In this study, we have synthesized and investigated binding property of a variety of WNA candidates. As a result, we have determined new potential analog, WNA-betaC for a CG interrupting site. It should be noted that the triplexes containing a WNA-betaT:TA or a WNA-betaC:CG base triplet have exhibited higher stability than the natural anti-parallel triplexes constructed of A:AT and G:GC triplets. The results that H-WNA-betaT lacking a benzene ring did not show stabilization effect to any base pairs have clearly indicated that a benzene ring plays a key role in stability of triplexes.

Base Sequence↗

PubMed1995

Perinatal lethal osteogenesis imperfecta.

Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes that encode the alpha 1(I) and alpha 2(I) chains of type I collagen, respectively. Point mutations resulting in the substitution of Gly residues in Gly-X-Y amino acid triplets of the triple helical domain of the alpha 1(I) or alpha 2(I) chains are the most frequent mutations. They interrupt the repetitive Gly-X-Y structure that is mandatory for the formation of a stable triple helix. Most babies have their own private de novo mutation. However, the recurrence rate is about 7% owing to germline mosaicism in one parent. The mutations act in a dominant negative manner as the mutant pro alpha chains are incorporated into type I procollagen molecules that also contain normal pro alpha chains. The abnormal molecules are poorly secreted, more susceptible to degradation, and impair the formation of the extracellular matrix. The collagen fibres are abnormally organised and mineralisation is impaired. The severity of the clinical phenotype appears to be related to the type of mutation, its location in the alpha chain, the surrounding amino acid sequences, and the level of expression of the mutant allele.

Female↗

PubMed1991

Complete primary structure of human collagen alpha 1 (V) chain.

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.

Amino Acid Sequence↗

PubMed1996

Specificity of antiparallel DNA triple helix formation.

We have used DNase I footprinting to examine the formation of antiparallel DNA triple helices on DNA fragments containing the homopurine target sites (GGA)2GGX(GGA)2GG.(CCT)2CCZ(CCT)2CC (where X.Z is each base pair in turn), with the GA- and GT-rich oligonucleotides, (GGA)2GGN(GGA)2GG and (GGT)2GGN(GGT)2GG (N = each base in turn). These were designed to form G.GC and A.AT or T.AT triplets with a central N.XZ mismatch, which should bind in an antiparallel orientation. We find that almost all combinations generate DNase I footprints at low micromolar concentrations. At each target site, the relative binding of the GA- and GT-containing oligonucleotides was not the same, suggesting that these two triplexes adopt different conformations. For a central GC base pair, the most stable complex is observed with a third strand generating a G.GC triplet as expected. A.GC is also stable, especially in the GT oligonucleotides. For a central AT base pair, all four bases form stable complexes though T.AT is favored for the GA-rich thirds strands and A.AT for the GT-rich strands. For a central CG base pair, the stable complexes are seen with third strands generating T.CG triplets, though A.CG and C.CG are stable with GT- and GA-containing oligonucleotides, respectively. C.TA is the best triplet at a central TA base pair. The third strands with central guanines avoided the formation of G.YR triplets on the fragments containing central pyrimidines, producing DNase I footprints which had slipped relative to the target site. These oligonucleotides bound at a different location, generating complexes containing 11 contiguous stable triplets at the 3'-end of the third strand. The results suggest rules for designing the best third strand oligonucleotides for targeting sequences in which homopurine tracts are interrupted by pyrimidines.

DNA↗

PubMed2000

Gene conversion (recombination) mediates expansions of CTG[middle dot]CAG repeats.

Genetic recombination is a robust mechanism for expanding CTG.CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468-23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome.

Base Sequence↗

PubMed2000

SCA8 CTG repeat: en masse contractions in sperm and intergenerational sequence changes may play a role in reduced penetrance.

We recently described an untranslated CTG expansion that causes a previously undescribed form of spinocerebellar ataxia (SCA8). The SCA8 CTG repeat is preceded by a polymorphic but stable CTA tract, with the configuration (CTA)(1-21)(CTG)(n). The CTG portion of the repeat is elongated on pathogenic alleles, which nearly always change in size when transmitted from generation to generation. To better understand the reduced penetrance and maternal penetrance bias associated with SCA8 we analyzed the sequence configurations and instability patterns of the CTG repeat in affected and unaffected family members. In contrast to other triplet repeat diseases, expanded alleles found in affected SCA8 individuals can have either a pure uninterrupted CTG repeat tract or an allele with one or more CCG, CTA, CTC, CCA or CTT interruptions. Surprisingly, we found six different sequence configurations of the CTG repeat on expanded alleles in a seven generation family. In two instances duplication of CCG interruptions occurred over a single generation and in other instances duplications that had occurred in different branches of the family could be inferred. We also evaluated SCA8 instability in sperm samples from individuals with expansions ranging in size from 80 to 800 repeats in blood. Surprisingly the SCA8 repeat tract in sperm underwent contractions, with nearly all of the resulting expanded alleles having repeat lengths of <100 CTGs, a size that is not often associated with disease. These en masse repeat contractions in sperm likely underlie the reduced penetrance associated with paternal transmission.

Alleles↗

PubMed1996

Regeneration of nerve fibres across a colonic anastomosis in the guinea-pig.

Resection and re-anastomosis of the bowel interrupts enteric neuronal pathways. The reestablishment of neuronal connections across a colonic anastomosis was studied using immunohistochemical, retrograde tracing and physiological techniques. In control guinea-pig proximal colon, retrograde labelling with 1,1'-didodecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) revealed that enteric neurons with anally-directed projections are more numerous and have longer axons than orally-projecting neurons. In resected bowel, up to 26 weeks after re-anastomosis, descending neuronal pathways were substantially interrupted. Immunohistochemical labelling of nerve fibres revealed that some enteric nerve fibres did regenerate across narrow regions of the anastomosis, growing preferentially in the oral to anal direction. However, nerve fibres immunoreactive for neurofilament protein triplet were substantially depleted in myenteric ganglia anal to the anastomosis, even after the longest recovery periods, demonstrating that axonal regrowth was limited. This was confirmed in retrograde tracing studies, as no nerve cell bodies oral to an anastomosis were labelled when DiI was placed on myenteric ganglia just anal to the anastomosis. Physiological studies confirmed that regrowth of nerve fibres across the anastomosis occurred and that it was asymmetric, as electrical stimulation led to aboral conduction across the anastomosis more reliably than oral conduction, as measured by circular muscle contraction. After resection and re-anastomosis of the colon, the disruption of neuronal pathways in the enteric nervous system was observed, with limited and preferential re-establishment of aborally-directed long connections.

Anastomosis, Surgical↗

PubMed1987

Translation framing code and frame-monitoring mechanism as suggested by the analysis of mRNA and 16 S rRNA nucleotide sequences.

Protein coding sequences carry an additional message in the form of a universal three-base periodical pattern (G-non-G-N)n, which is expressed as a strong preference for guanines in the first positions of the codons in mRNA and lack of guanines in the second positions. This periodicity appears immediately after the initiation codon and is maintained along the mRNA as far as the termination triplet, where it disappears abruptly. Known cases of ribosome slippage during translation (leaky frameshifts, out-of-frame gene fusion) are analyzed. At the sites of the slippage the G-periodical pattern is found to be interrupted. It reappears downstream from the slippage sites, in a new frame that corresponds to the new translation frame. This suggests that the (G-non-G-N)n pattern in the mRNA may be responsible for monitoring the correct reading frame during translation. Several sites with complementary C-periodical structure are found in the Escherichia coli 16 S rRNA sequence. Only three of them are exposed to various interactions at the surface of the small ribosomal subunit: (517)gcCagCagCegC, (1395)caCacCgcC and (1531)auCacCucC. A model of a frame-monitoring mechanism is suggested based on the weak complementarity of G-periodical mRNA to the C-periodical sites in the ribosomal RNA. The model is strongly supported by the fact that the hypothetical frame-monitoring sites in the 16 S rRNA that are derived from the nucleotide sequence analysis are also the only sites known to be actually involved or implicated in rRNA-mRNA interactions.

Base Sequence↗

PubMed1995

Genetic instability within monotonous runs of CpG sequences in Escherichia coli.

Genetic information can be altered by base substitutions, frameshift mutations, and addition or deletion of nucleotides. Deletions represent an important class of genetic aberration occurring at DNA sequences where it is often possible to predict the existence of intermediates of mutation. Instability within tracts of repetitive sequence have recently been associated with several genetic disorders, including the so-called triplet repeat diseases and certain forms of colorectal cancers. In Escherichia coli, (GpC)n repetitive sequences have been shown to be deletion prone, but the precise mechanism of this mutagenic pathway is still unknown. We show here that interrupting the monotony of the (GpC)n run with an ApT or a GpT dinucleotide decreases the rate of deletions within these sequences. On the other hand, introducing purine-pyrimidine alternating sequences beside the GpC insert results in an increased rate of deletion. Two pathways can be envisioned: (1) (GpC)n tracts can be seen as potential Z-forming DNA sequences, and this unusual DNA structure can be processed by an unknown cellular mechanism to give rise to the observed deletions and (2) (GpC)n monotonous runs can be considered as a succession of direct or palindromic repeats, allowing formation of DNA structures that are known to participate to frameshift mutagenesis. The results presented in this article are discussed in the light of these two alternative pathways.

Base Sequence↗

PubMed2003

Phase I-II study on isolation antiblastic fotemustine perfusion after dacarbazine chemosensitization for advanced melanoma of the extremities.

Isolation limb perfusion (ILP) is the treatment of choice for locally advanced limb melanoma. With melphalan, the referral drug, complete response (CR) is achieved in about 50% of patients, but significant local toxicity occurs in up to 30%. The aim of the present phase I-II study was to challenge fotemustine (F) in ILP after systemic chemosensitization with dacarbazine (DTIC), given its lower toxicity and greater efficacy, as reported in a previous pilot study. Eleven patients with locally advanced limb melanoma were subdivided into triplets, and given F ILP at escalating doses (starting from 25 mg/l) after intravenous administration of 500 mg/m2 DTIC. Acute and chronic locoregional and systemic toxicity, tumour response and clinical outcome were evaluated. Two patients in the first triplet had G3-G4 local toxicity, so that the scheduled F dosage was halved. At drug levels of 12.5, 15.6 and 18.2 mg/l, local toxicity decreased, but only one of eight patients showed CR. The trial was then interrupted due to the low tolerability and poor efficacy of this perfusion regimen. At present, F ILP after DTIC chemosensitization should not be recommended for the treatment of locally advanced limb melanoma.

Adult↗

PubMed2001

The comparative genomics of polyglutamine repeats: extreme differences in the codon organization of repeat-encoding regions between mammals and Drosophila.

Polyglutamine repeats within proteins are common in eukaryotes and are associated with neurological diseases in humans. Many are encoded by tandem repeats of the codon CAG that are likely to mutate primarily by replication slippage. However, a recent study in the yeast Saccharomyces cerevisiae has indicated that many others are encoded by mixtures of CAG and CAA which are less likely to undergo slippage. Here we attempt to estimate the proportions of polyglutamine repeats encoded by slippage-prone structures in species currently the subject of genome sequencing projects. We find a general excess over random expectation of polyglutamine repeats encoded by tandem repeats of codons. We nevertheless find many repeats encoded by nontandem codon structures. Mammals and Drosophila display extreme opposite patterns. Drosophila contains many proteins with polyglutamine tracts but these are generally encoded by interrupted structures. These structures may have been selected to be resistant to slippage. In contrast, mammals (humans and mice) have a high proportion of proteins in which repeats are encoded by tandem codon structures. In humans, these include most of the triplet expansion disease genes.

Animals↗

PubMed2000

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

Alleles↗

PubMed1995

Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene.

An unusual S1-nuclease sensitive microsatellite (STMS) has been found in the single copy, rat polymeric immunoglobulin receptor gene (PIGR) terminal exon. In Fisher rats, elements within or beyond the STMS are expressed variably in the 3' untranslated regions (3'UTRs) of two 'Groups' of PIGR-encoded hepatic mRNAs (pIg-R) during liver regeneration. STMS elements include neighboring constant regions (a 60-bp d[GA]-rich tract with a chi-like octamer, followed by 15 tandem d[GGA] repeats) that merge directly with 36 or 39 tandem d[GAA] repeats (Fisher or Wistar strains, respectively) interrupted by d[AA] between their 5th-6th repeat units. The Wistar STMS is flanked upstream by two regions of nearly contiguous d[CA] or d[CT] repeats in the 3' end of intron 8; and downstream, by a 283 bp 'unit' containing several inversions at its 5' end, and two polyadenylation signals at its 3' end. The 283 nt unit is expressed in Group 1 pIg-R mRNAs; but it is absent in the Group 2 family so that their GAA repeats merge with their poly A tails. In contrast to genomic sequence, GGA triplet repeats are amplified (n > or = 24-26), whereas GAA triplet repeats are truncated variably (n < or = 9-37) and expressed uninterruptedly in both mRNA Groups. These results suggest that 3' end processing of the rat PIGR gene may involve misalignment, slippage and premature termination of RNA polymerase II. The function of this unusual processing and possible roles of chi-like octamers in quiescent or extrahepatic tissues are discussed.

Animals↗

PubMed1992

Cloning, structural analysis, and expression of the glycogen phosphorylase-2 gene in Dictyostelium.

The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity. The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits. The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography. The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template. The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene. These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene. The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively. The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4. An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region. The CAA repeat was transcribed, as shown by the sequence of cDNA. Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively. Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development. Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development. However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide. Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.

Amino Acid Sequence↗

PubMed2002

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination.

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.

Crossing Over, Genetic↗

PubMed1994

DNA recognition by alternate strand triple helix formation: affinities of oligonucleotides for a site in the human p53 gene.

Duplex DNA recognition by oligonucleotide-directed triple helix formation is generally limited to homopurine target domains. Various approaches have been suggested for the relief of this constraint. Artificial DNA sequences have previously been used to show that adjacent homopurine domains on opposite DNA strands can be simultaneously recognized by oligonucleotide probes that switch triple helix recognition motifs between domains. Using assays of electrophoretic mobility and chemical protection, we have explored in detail whether such strategies are of benefit in designing high-affinity probes for a natural DNA sequence in the human p53 gene. This target site contains three adjacent, purine-rich domains on opposite DNA strands. Our results show that (i) a modest but statistically significant enhancement in affinity can be achieved for this sequence by designing an oligonucleotide that simultaneously recognizes all three purine domains, (ii) correction of a pyrimidine interruption in one purine domain does not dramatically alter this result, (iii) the relative energetic and structural contributions attributable to recognition of each purine domain can be assessed using probes with combinations of specific and nonspecific nucleotide sequences, and (iv) probe affinity is not correlated with the apparent number of base triplets for certain complexes. These data suggest that unfavorable free energy changes may be associated with alternation between triple helix motifs using existing strategies. In contrast to artificial DNA sequences optimized for this purpose, a substantial affinity enhancement was not observed using alternate strand DNA recognition at this natural target sequence. We therefore conclude that such enhancement is sequence dependent.

Base Sequence↗

PubMed1996

Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene.

The FMR-1 gene for the human fragile-chi syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5' non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG)n inserts (where n = 6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-chi syndrome.

Base Sequence↗

PubMed2004

Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation.

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.

Amino Acid Sequence↗