The limb-girdle muscular dystrophies (LGMD) are a collection of genetic diseases united in their phenotypical expression of pelvic and shoulder area weakness and wasting. More than 30 subtypes have been identified, five dominant and 26 recessive. The increase in the characterization of new genotypes in the family of LGMDs further adds to the heterogeneity of the disease. Meanwhile, better understanding of the phenotype led to the reconsideration of the disease definition, which resulted in eight old subtypes to be no longer recognized officially as LGMD and five new diseases to be added to the LGMD family. The unique variabilities of LGMD stem from genetic mutations, which then lead to protein and ultimately muscle dysfunction. Herein, we review the LGMD pathway, starting with the genetic mutations that encode proteins involved in muscle maintenance and repair, and including the genotype–phenotype relationship of the disease, the epidemiology, disease progression, burden of illness, and emerging treatments.

The plasminogen kringle 2 (K2HPg) and kringle 3 (K3HPg) modules occur in tandem within the polypeptide segment that affords the heavy chain of plasmin. The K2HPg and K3HPg are unique among the plasminogen kringle domains in that they also are linked to each other via the Cys169-Cys297 (Cys4 of K2HPg to Cys43 of K3HPg, kringle numbering convention) disulfide bridge, thus generating a K2HPg-K3HPg "supermodule". The kringle (2 + 3) sequence of human plasminogen (r-EE[K2HPgK3HPg]DS) was expressed in Escherichia coli, using an expression vector containing the phage T5 promoter/operator N250PSN250P29 and the codons for an N-terminal hexahistidine tag to ensure the isolation of the recombinant protein by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose under denaturing and reducing conditions. Kringle (2 + 3) was refolded in the presence of glutathione redox buffer. By taking advantage of the lysine affinity of kringle 2, the protein was purified by affinity chromatography on lysine-Bio-Gel. Recombinant kringle (2 + 3) was identified by amino acid composition, N-terminal sequence and mass determination. The 1H NMR spectrum shows that the intact r-K2HPgK3HPg is properly folded. By reference to spectra of the individual kringles, r-K2HPg and r-K3HPg, resonances of the K2HPg and K3HPg components in the spectrum of the intact r-K2HPgK3HPg can be readily distinguished. The strictly conserved Leu46 residue (kringle residue number convention) yields delta-methyl signals that are characteristic for K2HPg and K3HPg, exhibiting chemical shifts of -0.87 and -0.94 ppm, respectively, which are distinct from those of K1HPg, K4HPg, and K5HPg, (-1.04 to -1.05 ppm). Thus, the high-field Leu46 signals from K2HPg and K3HPg are well resolved from those of other kringles and can be identified unambiguously in spectra of the K1HPgK2HPgK3HPg elastolytic fragment of plasminogen as well as in spectra of Glu-plasminogen. Overall, r-K2HPgK3HPg exhibits broader resonance line widths than does the K1HPg component, consistent with a lesser mobility of the K2HPgK3HPg segment within the K1HPgK2HPgK3HPg fragment, a reflection of the extra structural constraint imposed by the disulfide bridge linking K2HPg to K3HPg. The ligand 6-aminohexanoic acid (6-AHA), which is known to interact with r-K2HPg but not with r-K3HPg, selectively perturbs K2 aromatic signals in the intact r-K2HPgK3HPg spectrum while leaving K3 resonances largely unaffected. Association constant (K(a)) values for 6-AHA determined from 1H NMR ligand titration experiments yield K(a) approximately 2.2 +/- 0.3 mM(-1) for the intact r-K2HPgK3HPg, comparable to K(a) approximately 2.3 +/- 0.2 mM(-1) determined for the isolated r-K2HPg, which demonstrates that the interactions of 6-AHA with the K2HPg ligand-binding site are not significantly affected by the neighboring K3HPg domain within the intact r-K2HPgK3HPg supermodule.

Recombinant BM 06.022 (M(r) 39,589) is a domain-deletion mutant of the human tissue-type plasminogen activator (tPA) structured by the kringle 2 and protease modules. Unfolding under various conditions was investigated via 1H-NMR spectroscopy by monitoring the well-resolved high-field methyl resonances at approximately -0.97 ppm (kringle 2) and approximately -0.29 and -0.54 ppm (protease). Reversible acid/base unfolding is manifest under low pH (< 4.8) conditions. It is observed that, relative to the protease, the kringle exhibits higher overall stability at low pH. At pH 4.6, BM 06.022 undergoes two distinct thermal melting transitions, at approximately 334 and approximately 352 K, assigned to an irreversible denaturation of the protease and a reversible unfolding of the kringle 2, respectively. Under the same conditions, the protease reacted with the active site inhibitor 1,5 dansyl-L-glutamylglycyl-L-arginine chloromethyl ketone (EGRck) exhibits a higher (approximately 10 K) thermal stability than the inhibitor-free protease. Upon acidification, the EGRck-modified protease unfolds irreversibly around pH 3.4. As exemplified by BM 06.022, a single-chain protein, as defined by continuity of the polypeptide backbone, can exhibit simultaneous folding reversibility and irreversibility for autonomous segments of the sequence. Conversion of the isolated (single-chain) protease or intact BM 06.022 to their catalytically active two-chain forms via plasminolytic cleavage of the Arg275-Ile276 peptide bond leaves the kringle 2 spectrum unaffected while perturbing the resolved high-field methyl resonances stemming from the protease. The latter also shift when the protease is reacted with EGRck, indicating that these signals are sensitive to events at the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Well-resolved high-field 1H NMR signals between -0.1 and -0.7 ppm afford convenient probes to monitor the conformational state of the tissue plasminogen activator (tPA) protease, modulated by covalent inhibitor binding or activation cleavage [Hu, C.-K., Kohnert, U., Wilhelm, O., Fischer, S., & Llinas, M. (1994) Biochemistry 33, 11760-11766]. We have investigated recombinant BM 06.022 (a domain-deletion variant mutant from Escherichia coli comprising the kringle 2 and protease modules) and protease constructs of tPA in both single-chain (sc) and two-chain (tc) forms. The two proteins were studied when confronted with the noncovalent (i.e., reversible) active site inhibitors benzamidine and a series of bisbenzamidine derivatives: 2,5-bis(4-amidinobenzylidene)cyclopentanone, 2,6-bis(4-amidinobenzylidene)cyclohexanone, 2,7-bis(4-amidinobenzylidene)cycloheptanone, and 2,8-bis(4-amidino- benzylidene)cyclooctanone. At pH* 4.6, the 1H NMR spectrum is sensitive to complexation of the protease module with the various effectors. The amplitude of the inhibitor-shifted resonances is more pronounced for the tc-protease than for the sc-protease, suggesting that access of inhibitors to the protease catalytic site is facilitated upon conversion to the tc form. The effects detected by the NMR spectrum suggest a biphasic process, involving stronger (primary) and weaker (secondary) bindings to a single protease active site. Binding to the protease module in tc-BM 06.022 essentially generates the same spectral characteristics as detected upon binding to the isolated tc-protease construct. In contrast, a negligible perturbation by the inhibitors is observed on the (sc) BM 06.022. Hence, in the intact BM 06.022 the kringle 2 is structurally coupled to the protease module thus interfering with inhibitor molecules from accessing the protease active site. These domain-domain interactions relax upon conversion to the catalytically active tc form, thus decoupling the kringle 2 from the protease module in BM 06.022 while simultaneously exposing the active site to become accessible to effectors or substrates.