Molecular characteristics and association analysis with litter size trait for porcine KLK7 gene.

Kallikrein-related peptidase 7(KLK7) is a tumor-related gene. In this study, the full-length coding sequence of porcine KLK7 gene was cloned through RT-PCR. Sequence analysis revealed that the pig KLK7 gene encodes a protein of 257 amino acids which has high homology with the KLK7 protein of six species: polar bear (95%), Weddell seal(94%), dog (92%), Pacific walrus (95%), domestic cat (92%), and Amur tiger (91%). This gene is structured in five exons and four introns as revealed by computer-assisted analysis. Phylogenetic analysis showed that the pig KLK7 gene has a closer genetic relationship with the KLK7 gene of a domestic cat. PCR-Alu I-RFLP was established to detect the GU373714:c.390 C > T substitution of porcine KLK7 gene and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size trait was assessed in Large White (n = 200) and Landrace (n = 200) pig populations and results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (p < 0.05). Therefore, KLK7 is also an important reproduction related gene.

Hemagglutinin Cleavability, Acid Stability, and Temperature Dependence Optimize Influenza B Virus for Replication in Human Airways.

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.

Kallikrein-related peptidases in cancers of gastrointestinal tract: an inside view of their role and clinical significance.

Human tissue kallikrein (KLK1) and is related peptidases (KLK2-KLK15) are a family of 15 homologous serine proteases, participating in numerous processes of normal physiology. Considering the irreversible impact of proteases on substrates, the tissue-dependent regulation of KLKs activity becomes crucial for their beneficial role in normal homeostasis. Moreover, KLKs expression is strongly regulated at the transcriptional and post-transcriptional level by steroid hormones and miRNAs, respectively. Deregulation of KLKs expression, secretion and/or activation has been observed in most human malignancies and there is a trend to identify their role in the multi-complex process of cancer development. The identification of extracellular matrix (ECM) proteins, cell-surface receptors, cell-surface adhesion molecules and growth factors among substrates, clearly support the driving role of KLK abnormal expression and function during tumorigenesis and cancer progression. KLKs have also clinical utility in cancer diagnosis and monitoring like KLK 3 (PSA) in prostate cancer. In this review, we tried to summarize the existing literature about the role of KLKs in gastrointestinal cancers as well as to emphasize their clinical significance for patients' prognosis.

Proteinase-activated receptors, targets for kallikrein signaling.

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).

An overview of the kallikrein gene families in humans and other species: emerging candidate tumour markers.

Kallikreins are serine proteases with diverse physiologic functions. They are represented by multigene families in many animal species, especially in rat and mouse. Recently, the human kallikrein gene family has been fully characterized and includes 15 members, tandemly localized on chromosome 19q13.4. A new definition has now been proposed for kallikreins, which is not based on function but, rather, on close proximity and structural similarities. In this review, we summarize available information about kallikreins in many animal species with special emphasis on human kallikreins. We discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and overview their evolutionary history. Kallikreins are expressed in a wide range of tissues including the salivary gland, endocrine or endocrine-related tissues such as testis, prostate, breast and endometrium and in the central nervous system. Most, if not all, genes are under steroid hormone regulation. Accumulating evidence indicates that kallikreins are involved in many pathologic conditions. Of special interest is the potential role of kallikreins in the central nervous system. In addition, many kallikreins seem to be candidate tumor markers for many malignancies, especially those of endocrine-related organs.

Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries.

To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.

Classification of serine proteases derived from steric comparisons of their active sites, part II: "Ser, His, Asp arrangements in proteolytic and nonproteolytic proteins".

The assignment of serine proteases to the families of (chymo)trypsins and subtilisins, respectively, is extended by including additional data from the Brookhaven Protein Data Bank. To better understand basic properties connected with this type of assignment the steric situation in the vicinity of the tetrad aminoacyl residues and atomic distances within the tetrads are considered. A new catalytic mechanism is suggested based on differences between tonin and kallikrein with respect to structure and reactivity of the catalytic tetrad. All protein structures available from the Brookhaven Protein Data Bank are analyzed with regard to the occurrence of Asp....His....Ser triads.

Classification of serine proteases derived from steric comparisons of their active sites.

The steric arrangements of the amino acyl residues in the catalytic triads and tetrads of the active site are compared with each other by means of systematic analysis of the conformation of the serine proteases stored in the Brookhaven Protein Data Bank. On this basis a differentiation between the representatives of the (chymo)trypsin family on the one hand and those of the subtilisin family on the other hand is found. The enzyme tonin distinguishes from representatives of the (chymo)trypsin family and should be classified to a new subclass of this family. Thermitase represents a new subclass of the subtilisins. The spatial orientation of the amino acyl residues of the active site of tonin suggests a new mechanism of enzyme catalysis that possibly also occurs in dipeptidyl peptidase IV.

Enhancement of lymphocyte proliferation by mouse glandular kallikrein.

Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.

Cleavage of human high-molecular weight kininogen by purified kallikreins and upon contact activation of plasma.

To study the digestion pattern of human high-molecular weight (mol wt) kininogen (HMWK) in plasma during contact activation we have prepared monoclonal antibodies (MoAbs) to the light-chain (LC) and the heavy-chain moiety of HMWK. One MoAb from each set was purified, and neither MoAb inhibited the clotting activity of HMWK. In enzyme-linked immunosorbent assay and immunoblotting experiments neither antibody bound to kininogen-deficient plasma. Digestion of purified HMWK with plasma kallikrein yielded, on reduced sodium dodecyl sulfate gels, two LC forms, at 62 and 49 kd, respectively. Digestion of HMWK with tissue kallikrein (TK) yielded mainly the 62-kd form. In immunoblot analyses of these digests, the anti-LC MoAb detected products at 62 and 49 kd respectively. With plasma kallikrein, the 62-kd species slowly shifted to 49 kd, and with TK, the 62-kd species accumulated with time. Anti-LC MoAb was also used as a probe in immunoblotting experiments to study the digestion pattern of HMWK in whole plasma activated with kaolin or dextran sulfate. In activated normal pooled plasma (NHP) and factor XI-deficient plasma, native HMWK (mol wt, 115 kd) was cleaved within five to ten minutes, and two LC forms at 62 and 49 kd were detected. In kaolin-activated prekallikrein (PK)-deficient plasma, the disappearance of the 115-kd form was relatively slow, and only the 62-kd form of LC was seen. HMWK was not cleaved when factor XII-deficient plasma was incubated with kaolin. LC-dependent coagulant activity paralleled the presence of LC bands seen in the immunoblots, and lower-mol wt fragments of LC were not identified. These data indicate that in activated NHP two forms of LC of HMWK (62 and 49 kd) are formed sequentially. Further, the LC-dependent coagulant activity remains detectable long enough to suggest that proteolytic inactivation of LC is too slow to be an important control mechanism.