Effect of SNPs on Creatine Kinase Structure and Function: Identifying Potential Molecular Mechanisms for Possible Creatine Kinase Deficiency Diseases

Abstract

Single-nucleotide polymorphisms (SNPs) are common genetic material changes that often occur naturally. SNPs can cause amino acid replacements that may lead to severe diseases, such as the well-known sickle-cell anemia. We constructed eight SNP mutants of human brain-type creatine kinase (CKB) based on bioinformatics predictions. The biochemical and biophysical characteristics of these SNP mutants were determined and compared to those of the wild-type creatine kinase to explore the potential molecular mechanisms of possible creatine kinase SNP-induced diseases. While the reactivation of six SNP mutants after heat shock dropped more than 45%, only three of them showed notable increases in ANS fluorescence intensity and decreases in catalytic efficiency. Among them, H26Y and P36T bind substrates as well as the wild-type form does, but the melting temperatures (Tm) dropped below body temperature, while the T59I mutant exhibited decreased catalytic activity that was most likely due to the much reduced binding affinity of this mutant for substrates. These findings indicate that SNPs such as H26Y, P36T and T59I have the potential to induce genetic diseases by different mechanisms.

Genetic variants in genes involved in creatine biosynthesis in patients with severe obesity or anorexia nervosa

“…Most of the identified non-synonymous variants in the genes of interest were predicted to impact the protein structure. Previously, alterations in the biochemical and biophysical properties of CKB caused by distinct genetic variations leading to amino acid substitutions have been analyzed ( Li et al, 2012 ). Eight non-synonymous variants in CKB were studied.…”

“…The results demonstrated that half of the variants’ amino acid substitutions affected the catalytic efficiency. For instance, the resultant amino acid exchange of rs13558 (p.Lys267GLu) increases the enzymatic activity by ∼ 30% compared to CKB containing the wild-type lysine ( Li et al, 2012 ). Further amino acid substitution due to other SNP alleles conversely impaired the catalytic efficiency.…”

“…Further amino acid substitution due to other SNP alleles conversely impaired the catalytic efficiency. In mice, some mutated alleles even implied reduced thermal stability below the physiological temperature of 37 °C ( Li et al, 2012 ). Li et al (2012) suspected that this impairment is related to thermoregulation in the double-KO mouse model ( Streijger et al, 2005 ; Streijger et al, 2009 ; Li et al, 2012 ).…”

“…In mice, some mutated alleles even implied reduced thermal stability below the physiological temperature of 37 °C ( Li et al, 2012 ). Li et al (2012) suspected that this impairment is related to thermoregulation in the double-KO mouse model ( Streijger et al, 2005 ; Streijger et al, 2009 ; Li et al, 2012 ). Our in silico analyses predominantly pointed to benign effects, while the majority of variants exhibited the potential to alter splicing products.…”

Improved Production of Highly Active and Pure Human Creatine Kinase MB

“…Asp-63 is among the residues that are predicted to be subjected to N-linked glycosylation. The amino acid is located in the 60-70 flexible loop [Bong et al, 2008;Li et al, 2012;McLeish and Kenyon, 2005]. Due to the high importance of the loop in catalysis, such glycosylation may decrease its flexibility and may reduce the activity.…”