Claude Lazure

GRANULINS, A NOVEL CLASS OF PEPTIDE FROM LEUKOCYTES

We report the isolation and characterization of a novel class of leukocyte peptides with possible cytokine-like activities which we call granulins. They are cystine-rich with molecular weights of approximately 6 Kda, except for granulin D, which appears to be a dimer. We present the sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes. A fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow. Rat granulin, and human granulin A, are closely related, showing that the granulin structures are highly conserved between species.

Circulating Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Regulates VLDLR Protein and Triglyceride Accumulation in Visceral Adipose Tissue

Objective—Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its gene is the third locus implicated in familial hypercholesterolemia. Herein, we investigated the role of PCSK9 in adipose tissue metabolism. Methods and Results—At 6 months of age, Pcsk9−/− mice accumulated ≈80% more visceral adipose tissue than wild-type mice. This was associated with adipocyte hypertrophy and increased in vivo fatty acid uptake and ex vivo triglyceride synthesis. Moreover, adipocyte hypertrophy was also observed in Pcsk9−/− Ldlr−/− mice, indicating that the LDLR is not implicated. Rather, we show here by immunohistochemistry that Pcsk9−/− males and females exhibit 4- and ≈40-fold higher cell surface levels of very-low-density lipoprotein receptor (VLDLR) in perigonadal depots, respectively. Expression of PCSK9 in the liver of Pcsk9−/− females reestablished both circulating PCSK9 and normal VLDLR levels. In contrast, specific inactivation of PCSK9 in the liver of wild-type females led to ≈50-fold higher levels of perigonadal VLDLR. Conclusion—In vivo, endogenous PCSK9 regulates VLDLR protein levels in adipose tissue. This regulation is achieved by circulating PCSK9 that originates entirely in the liver. PCSK9 is thus pivotal in fat metabolism: it maintains high circulating cholesterol levels via hepatic LDLR degradation, but it also limits visceral adipogenesis likely via adipose VLDLR regulation.

Identification of a biologically active circulating form of rat atrial natriuretic factor.

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.

Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator

Our work was undertaken to compare the relative efficiency of 2 purified prostatic kallikreins, namely, hK2 and prostate‐specific antigen (PSA or hK3), in the activation of single‐chain urokinase (scuPA). We found that hK2 converts scuPA into an active enzyme with an efficiency equal to approximately 1/50 that of plasmin. During the activation of scuPA by hK2, two fragments of 33 and 22 kDa were generated. The NH2‐terminal amino acid sequence of the 33 kDa fragment showed that hK2 cleaved scuPA between Lys158 and Ile159. In contrast to a previous report by another group, our purified hK3 preparation containing no trypsin‐like contaminants was totally unable to activate scuPA. Our results show that kallikrein hK2 has plasmin‐like activity and suggest that it could be the initiator of a proteolytic cascade leading to prostatic cancer invasion. Int. J.Cancer 71: 897‐899, 1997.

Gene Inactivation of Proprotein Convertase Subtilisin/Kexin Type 9 Reduces Atherosclerosis in Mice

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E–deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor–deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions— Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.Background —The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the LDL receptor (LDLR). PCSK9 gain-of-function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ~7-fold lower levels of LDL-cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results —We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E (apoE)-deficient and LDLR-deficient mouse models. Circulating cholesterol levels, FPLC profiles, aortic cholesteryl esters (CE) and plaque size were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (KO), normal (WT) or high (Tg) levels of PCSK9 were fed a 12 month-Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next, we generated apoE-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e) or high (Tg/e) levels of PCSK9. Following a 6 month-regular diet, KO/e mice showed a 39% reduction compared to WT/e mice in aortic CE accumulation, while Tg/e mice showed a 137% increase. Finally, LDLR-deficient mice expressing no (KO/L), normal (WT/L) or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited similar levels of plasma cholesterol and CE accumulation to WT/L, suggesting that PCSK9 modulates atherosclerosis mainly via the LDLR. Conclusions —Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is pro-atherogenic, while its absence is protective.

Gene Inactivation of PCSK9 Reduces Atherosclerosis in Mice

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E–deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor–deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions— Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.Background —The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the LDL receptor (LDLR). PCSK9 gain-of-function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ~7-fold lower levels of LDL-cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results —We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E (apoE)-deficient and LDLR-deficient mouse models. Circulating cholesterol levels, FPLC profiles, aortic cholesteryl esters (CE) and plaque size were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (KO), normal (WT) or high (Tg) levels of PCSK9 were fed a 12 month-Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next, we generated apoE-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e) or high (Tg/e) levels of PCSK9. Following a 6 month-regular diet, KO/e mice showed a 39% reduction compared to WT/e mice in aortic CE accumulation, while Tg/e mice showed a 137% increase. Finally, LDLR-deficient mice expressing no (KO/L), normal (WT/L) or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited similar levels of plasma cholesterol and CE accumulation to WT/L, suggesting that PCSK9 modulates atherosclerosis mainly via the LDLR. Conclusions —Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is pro-atherogenic, while its absence is protective.

NH2-terminal fragment of rat pro-atrial natriuretic factor in the circulation: Identification, radioimmunoassay and half-life

A radioimmunoassay was developed to measure the NH2-terminal counterpart of rat pro-atrial natriuretic factor (pro-ANF) in plasma. Synthetic rat ANF (Asp 11-Ala 37) coupled to bovine serum albumin was used to immunize New Zealand rabbits. The antiserum demonstrated good immunoreactivity towards rat ANF (Asn 1-Arg 98), (Asn 1-Tyr 126), (Asp 11-Ala 37) and even human ANF (Asn 1-Ser 30). The standard curve had an ED80 of 9.5 +/- 2.5 and ED50 of 44.0 +/- 10.5 fmol/tube. Immunoreactive ANF NH2-terminal peptide was measured directly in rat plasma without prior extraction. In fact, extraction of ANF NH2-terminal from plasma by C18 silica gel chromatography revealed inconsistent recovery and a lack of parallelism. Morphine (0.75 mg/100 g), chosen to elicit increased ANF (Ser 99-Tyr 126) secretion, elevated its plasma concentration from 54.1 +/- 3.2 to 190.8 +/- 55.8 fmol/ml after 20 min. At the same time, the immunoreactive NH2-terminal fragment rose from 378 +/- 16 to 1181 +/- 201 fmol/ml. The identity of this immunoreactive material was verified following affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC) of plasma from morphine-treated rats. Molecular sieving and amino acid sequencing demonstrated that it appears to be consistent with or identical to rat ANF (Asn 1-Arg 98). The disappearance rate of ANF (Asn 1-Arg 98) was studied by injecting radioactive material into anesthetized rats. The exponential decay was analyzed by a two-compartment model in which the fast and slow components had a half-life of 2.5 +/- 0.3 and 54.8 +/- 3.9 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma

To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.

Primary structure of a high Mr form of rat atrial natriuretic factor

During the purification of rat atrial natriuretic factor (ANF), low, intermediate and high M r forms were observed. In this report we describe the purification and amino acid sequence of a 73 residue peptide containing at its C‐terminus the previously sequenced 33 amino acid ANF peptide. The cleabage necessary to produce the 33 amino acid ANF from the 73 amino acid precursor occurs at a LeuLeu bond. We also report the amino acid composition of an even longer form of ANF containing about 103 residues, in which the extension is amino terminal to the 73 peptide. A computer data bank search showed that the determined sequence is a novel one and is not homologous to any known proteins or segment thereof. The natriuretic activity of the 73 amino acid form when compared to that of a synthetic ANF peptide, comprising the sequence of the last 26 amino acids of ANF, was found to be slightly lower.

Isolation and characterization of gelatin-binding proteins from goat seminal plasma

A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hens egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.