We have recently developed a new target plate (BLOTCHIP®) for MALDI-MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy-induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC-MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen-1 (three peptides), fibrinogen-α, complement component C4-A/B, α-2-HS-glycoprotein and inter-α-trypsin inhibitor heavy chain H4. 2-D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP® analysis can be applied for discovery study of PIH biomarker candidates.
Abstract. To investigate molecular effects of anti-sperm autoantibodies on fertilization, we previously established antimouse sperm-head auto-monoclonal antibodies (mAbs). Among the mAbs established, one mAb (named Ts4) recognized the sugar moiety of TEX101, a germ cell-marker glycoprotein. In the present study, we examined the immunoreactivity of Ts4 in mouse spermatozoa and fertilized eggs during early embryogenesis to clarify the distribution of the Ts4-reactive antigen in the fertilization process. Similar to TES101 mAb (a specific probe for TEX101), immunopositive staining of Ts4 was observed on spermatocytes, spermatids and spermatozoa within the testis. In contrast to the results obtained with TES101 mAb, Ts4 reacted with the sperm acrosomal region within the cauda epididymis. A Western blot analysis of epididymal sperm extract revealed that Ts4 mainly detected two bands between 100 and 150 kDa, while Ts4 faintly detected a band corresponding to TEX101 at 38 kDa. In addition, Ts4-reactive molecules were observed in the growing early embryo after fertilization. Since Ts4-reactive antigen, potentially a carbohydrate chain, is only observed in reproduction-related areas such as the testis, epididymal sperm-head and early embryo, it is expected to have an effect on fertilization. Therefore, additional studies of this antigen may elucidate the molecular mechanisms underlying the reproductive process. nti-sperm autoantibodies are present in the sera of some infertile patients [1]; however, the precise mechanism underlying the induction of anti-sperm antibodies remains unclear. One leading hypothesis suggests that cross-reactive immune responses against external antigens (e.g., bacterial or viral infections) may induce an immune response against sperm antigens [2]. Based on this hypothesis, we developed several anti-sperm auto-monoclonal antibodies (mAbs) using spleen cells from aged mice (over 1 year old) kept under normal conditions. To identify hybridoma cell lines that secrete anti-sperm autoantibodies, culture media from each hybridoma line were screened for antibody activity against mouse epididymal spermatozoa using ELISA and immunofluorescence tests (Fig.1). To pinpoint the specific antigens recognized by the anti-sperm auto-mAbs, we performed a micro amino acid analysis against testicular proteins that showed an immunopositive reaction for the mAbs using a two-dimensional SDS-PAGE system (Fig.1). Among the mAbs produced, one specific mAb (named Ts4) reacted with a protein that possessed an N-terminus of TYC-QVSQTLSLEDD [3]; this sequence shares 100% sequence identity with the potential N-terminal sequence of TEX10126-39 [4]. TEX101, a highly N-glycosylated glycosylphosphatidylinositol (GPI)-anchored glycoprotein [5], was originally identified as a protein containing the antigen epitope for a specific mAb, called TES101, produced by immunizing female mice with an allogenic testicular homogenate [4]. Previous studies by our research group have demonstrated that TEX101 is a unique germ cell marker that is ...
TEX101, a member of the Ly-6/urokinase-type plasminogen activator receptor (LU)-family we previously identified, is a germ cell-marker glycoprotein. To date, it is reported that some members of the protein-family are overexpressed in a variety of cancer tissues. We previously reported Ly6k, a member of the LU-family, as an association molecule with TEX101 in murine male germ cells. LY6K (a human homologue of Ly6k) is overexpressed in head and neck squamous cell carcinomas (HNSCC). These facts led us to speculate that TEX101 may also exist in HNSCC, like LY6K. Using an anti-human TEX101 polyclonal antibody (pAb) established, we examined the expression of TEX101 protein in cancer tissues by immunohistochemical analysis. TEX101 was detected in the cancer cells of some tissue specimens from patients with HNSCC, whereas the normal squamous epithelium was immunonegative. The TEX101 protein was detected in cancer cells from 54 out of 64 (80.6%) patients with HNSCC. The rate of lymph nodes metastasis tends to be low in TEX101-positive patients, compared to patients with weakly positive and negative expression of TEX101. The present results imply that TEX101 is a novel cancer-related protein and may be useful as a marker for prognosis/diagnosis of HNSCC.
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