Ian D. Walker

SP-40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.

We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 micrograms/ml) in normal human serum. SP-40,40 is deposited in the renal glomeruli of patients with glomerulonephritis but is not found in normal glomeruli. The protein is a heterodimeric structure of relative molecular mass 80 kD, both chains of which are of a similar size (40 kD). The amino-terminal sequences of both chains are unrelated to one another and possess no significant homology to any known protein sequence. The tissue distribution of SP-40,40 closely resembles that of the terminal complement components and its physicochemical properties are similar to, but distinct from, those of the S protein of complement. We have identified SP-40,40 in the SC5b-9 complex of complement and have demonstrated incorporation of labeled SP-40,40 into this complex. These data suggest that SP-40,40 is an additional component of SC5b-9.

Molecular cloning and characterization of the novel, human complement-associated protein, SP-40,40: a link between the complement and reproductive systems.

The cDNA sequence encoding the human complement‐associated protein, SP‐40,40, is reported. The two chains of SP‐40,40 are coded in a single open reading frame on the same mRNA molecule, indicating the existence of a biosynthetic precursor protein which matures post‐synthetically by the proteolysis of at least one peptide bond. The precursor is preceded by a signal sequence for vectorial export and contains six N‐linked glycosylation sites distributed equally between the two chains of the structure. The sequence of the SP‐40,40 precursor bears a 77% identity to a rat sulphated glycoprotein‐2 (SGP‐2) which is the major secreted product of Sertoli cells. The presence of SP‐40,40 within human seminal plasma at levels comparable to those in serum was demonstrated, indicating that SP‐40,40 and SGP‐2 are serum and seminal forms of the same protein. A sequence of 23 amino acids within the beta‐chain of SP‐40,40 exhibited significant homology to corresponding segments located within complement components C7, C8 and C9. The short cysteine‐containing motif represented the only evidence of a possible vestigial relationship between SP‐40,40 and other complement components. The precise role of SP‐40,40 is not known in either blood or semen but the present findings document an intriguing link between the immune and the reproductive systems.

Human seminal clusterin (SP-40,40). Isolation and characterization.

Molecular cloning of the human complement inhibitor SP-40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis. The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles. Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility.

The organisation of the multigene family which encodes the major cell surface protein, pMGA, of Mycoplasma gallisepticum

The genome of the avian pathogen Mycoplasma gallisepticum contains a number of related genes for putative adhesion molecules (pMGA). Cloning and sequence analysis of several pMGA genes suggested that all of them might be transcriptionally and translationally functional. Analysis of the gene sequence encoding the sole pMGA variant expressed in vitro in the S6 strain (pMGA1.1) revealed no unambiguous feature that could account for its unique expression. It is estimated that the pMGA gene family may contain up to 50 members, and its possible role is discussed herein.

SP-40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges

SP‐40,40 is a two‐chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (α—β) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established in this study for SP‐40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP‐40,40 and other multi‐chain proteins containing disulphide bonds.

pMGA Phenotypic Variation in Mycoplasma gallisepticum Occurs In Vivo and Is Mediated by Trinucleotide Repeat Length Variation

ABSTRACT Chickens were infected with a pathogenic strain of Mycoplasma gallisepticum, and the expression of pMGA, the major surface protein, was inferred by examination of colonies from ex vivo cells. Within 2 days postinfection, 40% of cells had ceased the expression of the original pMGA surface protein (pMGA1.1), and by day 6, the majority of recovered cells were in this category. The switch in pMGA phenotype which had occurred in vivo was reversible, since most colonies produced from ex vivo progenitors exhibited frequent pMGA1.1+sectors. After prolonged in vivo habitation, increasing proportions of recovered cells gave rise to variant pMGA colonies which had switched from the expression of pMGA1.1 to another gene, pMGA1.2, concomitant with the acquisition of a (GAA)12 motif 5′ to its promoter. Collectively, the results suggest that changes in M. gallisepticum pMGA gene expression in vivo are normal, common, and possibly obligate events for successful colonization of the host. Surprisingly, the initial cessation of pMGA1.1 expression occurred in the absence of detectable pMGA antibodies and seemed to precede the adaptive immune response.

Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6

A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell.

Ly Antigens Associated with T Cell Recognition and Effector Function

The mechanisms involved in acquisition and expression of T cell immunity are being extensively investigated in many laboratories. Monoclonal antibodies occupy a central role in the study of T cell immunity at both cellular and molecular levels and have facilitated the identification of the elements which figure in and result from the process of T cell differentiation. The purpose of this review is to identify key structures known to participate in T cell effector function and to describe some of our structural studies on 3 of these antigens, Ly-2/3, T8 and Ly-15.

Fractionation of detergent lysates of cells by ammonium sulphate-induced phase separation

A procedure is described for fractionating detergent lysates of cells based on the ability of (NH4)2SO4 to induce phase separation of detergents such as Triton X-100, sodium deoxycholate, and sodium cholate, into detergent-rich and detergent-depleted phases. An analysis of six murine lymphocyte cell surface molecules revealed that the partitioning in Triton X-100 of each molecule was highly dependent upon the (NH4)2SO4 concentration, each antigen partitioning into the detergent-rich phase at a defined salt concentration. In contrast, none of the six molecules appeared in the detergent-rich phase of a Triton X-114 phase separation, even though two of the molecules, namely Ly-2/3 and L3T4, are well-characterized integral membrane proteins. It was also observed that (NH4)2SO4 resulted in the partitioning of many nonmembrane proteins into the detergent-rich phase, indicating that the procedure can be used to fractionate all cellular proteins. By judicious choice of (NH4)2SO4 concentrations, precipitation of cellular proteins at two different (NH4)2SO4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.

Use of Click Chemistry to Define the Substrate Specificity of Leishmania β-1,2-Mannosyltransferases

Leishmania spp. are human pathogens that utilize a novel β‐1,2‐mannan as their major carbohydrate reserve material. We describe a new approach that combines traditional substrate‐modification methods and “click chemistry” to assemble a library of modified substrates that were used to qualitatively define the substrate tolerance of the Leishmania β‐1,2‐mannosyltransferases responsible for β‐1,2‐mannan biosynthesis. The library was assembled by using the highly selective copper(I)‐catalysed cycloaddition reaction of azides and alkynes to couple an assortment of azide‐ and alkyne‐functionalized small molecules with complementary alkyne‐ and azide‐functionalized mannose derivatives. All mannose derivatives with α‐orientated substituents on the anomeric carbon were found to act as substrates when incubated with a Leishmania mexicana particulate fraction containing GDP‐mannose. In contrast, 6‐substituted mannose derivatives were not substrates. Representative products formed from the library compounds were analysed by mass spectrometry, methylation linkage analysis and β‐mannosidase digestions and showed extension with up to four β‐1,2‐linked mannosyl residues. This work provides insights into the substrate specificity of this new class of glycosyltransferases that can be applied to the development of highly specific tools and inhibitors for their study.