Alpha1 Region Research Articles

Gem Stabilizes Voltage-Gated Calcium Channels in the Inactivated State: Implications for Human Disease

RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that often colocalize with and strongly inhibit high-voltage activated (HVA) calcium channels (L-, N-, P/Q- and R-type). The mechanism of inhibition is largely unclear. The pore-forming alpha-1 subunit of HVA calcium channels requires the beta subunit for cell surface trafficking and gating. We previously showed direct binding between alpha-1 and Gem and obtained a Gem-insensitive chimeric P/Q alpha-1containing the IIS1-S3 region of a Gem-resistant, low-voltage activated T-type channel (Fan et al., PNAS, 2010). In this study, we investigated the cause of this insensitivity. Since the IIS1-S3 region of P/Q alpha-1 has too few cytosolic amino acids to be a Gem binding site, and since we had identified a different alpha-1 region that binds Gem, we focused on altered biophysical properties of the chimera. We observed that the chimeric channel inactivated much slower than WT P/Q channel did; we therefore examined whether slow inactivation was responsible for Gem insensitivity. Mutating residues known to increase channel inactivation enhanced Gem inhibition, whereas mutating residues known to slow channel inactivation weakened or abolished Gem inhibition. Replacing beta3 with beta2a, an auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition. Furthermore, recovery from inactivation of WT P/Q channels in the presence of Gem was markedly slower than without Gem. Thus, Gem appeared to stabilize the channel in the inactivated state. These results shed light on a new factor - altered regulation by RGK proteins - that may contribute to HVA calcium channel-related channelopathies. Indeed, we found that familial hemiplegic migraine mutations that speed P/Q channel inactivation strengthened Gem inhibition, whereas Timothy syndrome mutations that slow channel inactivation greatly weakened Gem inhibition.

Polymorphism of the human alpha1 immunoglobulin gene 3' enhancer hs1,2 and its relation to gene expression.

We studied the hs1,2 transcriptional enhancer identified downstream of the human alpha1 gene of the immunoglobulin H (IgH) locus, for which two different allelic configurations (a and b) were previously reported by Southern blotting. By using a polymerase chain reaction (PCR) method we amplified minisatellites within the hs1,2 core enhancer, with variable numbers of tandem repeats (VNTR) defining three 'PCR alleles' alpha1A, alpha1B and alpha1C (including one, two and three repeats, respectively). Five different alpha1 h1,2 genotypes were encountered in a population of 513 donors, representing 13.8, 34.5, 49.7, 1.3 and 0.6% for the AA, BB, AB, AC and BC genotypes, respectively. Luciferase assays showed that increasing the number of minisatellites increased the transcriptional strength of the alpha1 hs1,2 enhancer. Simultaneous determination of Southern blot alleles and VNTR alleles only showed a partial linkage between both types of polymorphism, altogether defining at least six different allelic forms of the 3'alpha1 region. In conclusion, the present study further demonstrates the genetic instability of the 3'alpha region, for which multiple alleles have been generated through inversions and internal deletions and/or duplications. This study also strengthens the hypothesis that the polymorphism at the IgH 3' regulatory region of the alpha1 gene could play a role in the outcome of diseases involving immunoglobulin secretion.

Combinatorial Analysis of the Structural Requirements of theEscherichia coli Hemolysin Signal Sequence

We have investigated the substrate specificity of the Escherichia coli hemolysin transporter system. Translocation of hemolysin is dependent on a C-terminal signal sequence located within the last 60 amino acids of this protein. Previous comparative studies of the signal sequence have revealed a conserved helix(alpha1)-linker-helix(alpha2) motif, suggesting that secondary structure is important for transport. In this study, we generated three random libraries in the alpha1, linker, and alpha2 regions, as well as an alpha1-amphiphilic helical library to identify features buried within the structural motif that contribute to transport. Combinatorial variants were generated by altering the primary sequence of specific regions, and correlation between the genotype and phenotype of the mutant populations allowed us to objectively identify any functional features involved. It was found that the alpha1-amphiphilic helix and the linker are both important for function. To our surprise, the second helix of the conserved structural motif was not essential for transport. The finding that a predicted amphiphilic helix and hydrophobicity, rather than primary sequence, contribute to transport in the alpha1 region allows us to speculate on the mechanism of multiple substrate recognition. This may have implications for understanding the broad substrate specificity common among other ATP-binding cassette transporters.

The effects of exogenous free fatty acids on lipoprotein migration in serum high-resolution electrophoresis: addition of free fatty acids improves visualization of normal and abnormal alpha1-antitrypsin.

Abnormalities in the alpha1 region are occasionally difficult to interpret on high-resolution electrophoresis (HRE) gels because alpha1-antitrypsin (A1AT) can be obscured by elevated levels of lipoprotein. The addition of saturated free fatty acids (SFFAs) to serum samples causes a selective anodal migration of lipoproteins when examined by HRE. This "clearing" of the alpha1 region improves visualization of A1AT. In this study, we evaluated 6- to 24-carbon SFFAs for their ability to cause anodal migration of serum lipoproteins; identified the optimal SFFA and determined its effects on other proteins; and applied the SFFA to problematic serum samples, including those containing dense alpha lipoprotein regions. When added to serum samples, a mixture of medium-chain (12-18 carbon) SFFAs caused a dose-dependent anodal migration of the alpha and beta lipoproteins and albumin. Lauric acid (C(12:0); 3.2-6.5 mmol/L) caused an optimal effect--maximal anodal migration of alpha lipoproteins and clearing of the alpha1 region, facilitating inspection of that area for A1AT variants. At the optimal C(12:0) concentration, the migration and resolution of A1AT were minimally affected, anodal slurring of beta lipoproteins and albumin were slight, and there was no effect on interpretation of monoclonal proteins. At higher concentrations, C(12:0) increased anodal migration of beta lipoproteins and decreased the resolution of A1AT. C(12:0) at 3.2 mmol/L in serum samples of heparinized patients showed an exaggerated effect similar to higher concentrations of C(12:0) in nonheparinized serum samples. Hyperbilirubinemia did not alter the effect of C(12:0) on serum proteins. C(12:0) treatment of serum samples displaying dense alpha lipoprotein bands on HRE dramatically improved visualization of A1AT. We report the incidental identification of A1AT variants in two such samples treated with C(12:0). The addition of C(12:0) to serum samples markedly improves visualization of normal and abnormal A1AT in the alpha1 region in HRE.

Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide.

Peptides derived from the alpha 1-region of the murine H-2Dk molecule enhance glucose uptake in rat adipose cells above the maximum obtained with insulin stimulation alone (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now describe that epidermal growth factor (EGF) in combination with the same peptides, Dk-(61-85) and Dk-(62-85), stimulates cellular glucose uptake 5-7 times over the basal level, i.e. to 30-50% of the maximal insulin effect. EGF alone increased glucose uptake by only approximately 50% above basal and the peptide alone by 100% above basal. Maximal effect of EGF and peptide was reached in 10-20 min with 30 microM peptide (EC50 10-15 microM) and 50 nM EGF (EC50 1-2 nM). The effect of EGF and peptide on glucose uptake was additive to that of insulin and peptide until the maximal level attained with insulin and peptide was reached. The combined effect of EGF plus peptide on glucose transport was associated with a recruitment of GLUT4 molecules to the plasma membrane. However, the phosphatidylinositol (PI) kinase which is activated by insulin was not activated by EGF plus peptide. Thus, the effect of EGF plus peptide on glucose uptake seems independent of the activity status of the insulin receptor. 125I-Labeled EGF bound specifically to rat adipose cells with an apparent affinity of approximately 2 nM and Bmax approximately 5 x 10(3). However, the major histocompatibility complex (MHC) peptides did not affect EGF-stimulated internalization of EGF receptor, in contrast to their effect on the insulin receptors. Transforming growth factor alpha had an effect similar to EGF on glucose uptake. Three other peptides derived from other parts of murine MHC class I had no effect on glucose uptake in combination with EGF. Thus, EGF in combination with certain MHC class I-derived peptides is insulinomimetic concerning glucose transport and this effect is independent of the insulin receptor activity.

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent.

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.

Protease Inhibitors in Human Milk

Protease inhibitors (inhibiting trypsin, chymotrypsin, and elastase) were demonstrated in human milk from birth to 4 months after delivery. No pepsin inhibitor was found. The protease inhibitors were localized in the alpha 1-region--inhibiting trypsin, chymotrypsin, and elastase--and in a more cathodal region--inhibiting chymotrypsin--in agarose gel electrophoresis of human milk. alpha 1-antitrypsin and antichymotrypsin were demonstrated by crossed immunoelectrophoresis. Electroimmunoassay showed the concentration of alpha 1-antitrypsin in 1st day milk to be 10.9% and the concentration of antichymotrypsin was 116% of that of adult serum. The concentrations decreased during the 1st wk; from 1 wk to 4 months they were 1.6% for alpha 1-antitrypsin and 3.8% for antichymotrypsin of those of adult serum. One ml of human milk from the first 3 days inhibits, by enzymatic methods, 0-150 microgram (mean value, 70 microgram) trypsin. Milk from 1 week after delivery and later inhibits 0-65 microgram (mean value, 38 microgram) trypsin per ml.

Unique human glycoprotein, alpha1-microglycoprotein: isolation from the urine of a cancer patient and its characterization.

A human glycoprotein was isolated from the urine of a patient with plasma cell leukemia. It appears pure and homogeneous when examined by immunoelectrophoresis, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, gel filtration in 6 M guanidine hydrochloride (Gdn.HCl), and NH2-terminal amino acid sequence analysis. It has a brown color due to a tightly (most likely covalently) bound chromophore group(s) and migrates to the alpha1 region in immunoelectrophoresis. A molecular weight (mol wt) of 27 000 was obtained for the glycoprotein by gel filtration in 6 M Gdn.HCl. Its approximate mol wt determined by Na-DodSO4-polyacrylamide gel electrophoresis is 29 000 on 5% and 7.5% and 10% gels. Amino acid and hexosamine analyses showed that it is a glycoprotein and indicated that it contains four half-cystine residues per molecule. Based on the above observations we designated it "alpha1-microglycoprotein" (alpha1-MGP). Isoelectric focusing of alpha1-MGP showed a significant charge heterogeneity, although only a single NH2-terminal amino acid sequence was obtained for alpha1-MGP, i.e., Gly-Pro-Val-Pro-( )-Pro-Pro-Asx-Asx-Ile-Glx-Val-Glx-Glx-Asx-Phe-Phe-Ile-(Ser or Ala)-Arg. The alpha1-MGP was found in significant concentrations in the urine of many patients with neoplastic diseases.

The “Post-heparin Pattern” in Routine Serum Protein Immunoelectrophoresis

For some time following intravenous administration of heparin to non-fasting normal subjects, routine immunoelectrophoretic preparations show characteristic changes in serum lipoproteins. These alterations are of two types: (1) marked anodal mobility increase, the alpha-2 (beta) component locating in the alpha-1 region while the alpha-1 appears as a pre-albumin; (2) conformational alterations of the immunoprecipitin arcs comprising elongation of the alpha-2 (beta) arc, and splitting of the alpha-1 arc into two distinct entities. Familiarity with these patterns should minimize the possibility of interpretative errors with specimens from patients receiving heparin.

Anomalous Serum Protein in Hepatocarcinoma and Hepatic Metastases

An anomalous protein in the sera of patients affected by hepatic metastases or hepatocarcinoma can be readily demonstrated by “disk” electrophoresis, in which it migrates to the alpha1 region. The protein has not been found in sera of the newborn or in other pathologic conditions; it does not have antigenic properties. The hepatic origin of the protein and its diagnostic value in identifying primary and secondary hepatic neoplasia are discussed.